DNA-based digital information storage with sidewall electrodes

ABSTRACT

Provided herein are compositions, devices, systems and methods for generation and use of biomolecule-based information for storage. Further provided are devices-having addressable electrodes controlling polynucleotide synthesis (deprotection, extension, or cleavage, etc.) The compositions, devices, systems and methods described herein provide improved storage, density, and retrieval of biomolecule-based information.

CROSS-REFERENCE

This application claims the benefit of U.S. provisional patentapplication No. 62/613,728 filed on Jan. 4, 2018; U.S. provisionalpatent application No. 62/617,067 filed on Jan. 12, 2018; and U.S.provisional patent application No. 62/650,231 filed on Mar. 29, 2018,all of which are incorporated herein by reference in their entirety.

BACKGROUND

Biomolecule based information storage systems, e.g., DNA-based, have alarge storage capacity and stability over time. However, there is a needfor scalable, automated, highly accurate and highly efficient systemsfor generating biomolecules for information storage.

BRIEF SUMMARY

Provided herein are devices for storing information, comprising: a solidsupport, wherein the solid support comprises a plurality of wells,wherein each of the wells comprises an addressable locus comprising: asynthesis surface located in a bottom region of each of the wells; abottom electrode in addressable communication with the synthesissurface; and at least one sidewall electrode located on a sidewall ofeach of the wells, wherein the at least one sidewall electrode is 50 nmto 200 nm from the bottom region. Further provided herein are deviceswherein the solid support comprises addressable loci at a density of atleast 100×10⁶ addressable loci per cm². Further provided herein aredevices wherein the solid support comprises addressable loci at adensity of 100×10⁶ to 100×10⁷ addressable loci per cm². Further providedherein are devices wherein the addressable locus comprises a diameter upto about 750 nm. Further provided herein are devices wherein each of thewells comprises a depth up to about 1000 nm. Further provided herein aredevices wherein each of the wells comprises a depth of 100 nm to 1000nm. Further provided herein are devices wherein each of the wellscomprises a longest cross-sectional diameter of 100 nm to 800 nm.Further provided herein are devices wherein each of the wells iscylindrical. Further provided herein are devices wherein the bottomelectrode comprises a largest cross-sectional area of 10⁴ nm² to 10⁵nm². Further provided herein are devices wherein the at least onesidewall electrode is 50 nm to 200 nm from the bottom region. Furtherprovided herein are devices wherein the at least one sidewall electrodecomprises a height of 5 nm to 25 nm. Further provided herein are devicescomprising at least two sidewall electrodes. Further provided herein aredevices wherein the at least one sidewall electrode and the bottomelectrode are independently addressable.

Provided herein are devices for storing information, comprising: a solidsupport, wherein the solid support comprises a plurality of wells,wherein each of the wells comprises an addressable locus comprising: asynthesis surface located in a bottom region of each of the wells; abottom electrode in addressable communication with the synthesissurface; at least one sidewall electrode located on a sidewall of eachof the wells, wherein the synthesis surface at each addressable locuscomprises at least one polynucleotide extending from the synthesissurface, and wherein the polynucleotides comprising different sequenceson the solid support are present at a density of at least 100×10⁶polynucleotides per cm². Further provided herein are devices wherein thesolid support comprises polynucleotides of different sequences at adensity of at least 100×10⁷ polynucleotides per cm². Further providedherein are devices wherein the solid support comprises addressable lociat a density of 100×10⁶ to 100×10⁷ polynucleotides per cm². Furtherprovided herein are devices wherein each of the wells comprises a depthup to about 1000 nm. Further provided herein are devices wherein each ofthe wells comprises a depth of 100 nm to 1000 nm. Further providedherein are devices wherein the addressable locus comprises a diameter upto about 750 nm. Further provided herein are devices wherein each of thewells comprises a longest cross-sectional diameter of 100 nm to 800 nm.Further provided herein are devices wherein each of the wells iscylindrical. Further provided herein are devices wherein the bottomelectrode comprises a largest cross-sectional area of 10⁴ nm² to 10⁵nm². Further provided herein are devices wherein the at least onesidewall electrode is 50 nm to 200 nm from the bottom region. Furtherprovided herein are devices wherein the at least one sidewall electrodecomprises a height of 5 nm to 25 nm. Further provided herein are devicescomprising at least two sidewall electrodes. Further provided herein aredevices wherein the at least one sidewall electrode and the baseelectrode are independently addressable.

Provided herein are methods for storing information, comprising: (a)providing a device described herein; (b) providing instructions forpolynucleotide synthesis; (c) depositing at least one nucleoside on thesynthesis surface, wherein the at least one nucleoside couples to apolynucleotide attached to the synthesis surface; and (d) repeating stepc) to synthesize a plurality of polynucleotides on the synthesissurface, wherein the instructions comprise at least one sequenceencoding for the plurality of polynucleotides. Further provided hereinare methods further comprising cleaving at least one polynucleotide fromthe surface, wherein the polynucleotide is dissolved in a droplet.Further provided herein are methods further comprising sequencing atleast one polynucleotide from the surface. Further provided herein aremethods wherein the nucleoside comprises a nucleoside phosphoramidite.Further provided herein are methods further comprising a cleavage step,wherein the cleavage step comprises applying an electrical potential tothe bottom electrode to generate a cleavage reagent. Further providedherein are methods wherein the method further comprises drying thesurface. Further provided herein are methods further comprising washingthe nucleosides away from the surface. Further provided herein aremethods wherein the method further comprises a capping step. Furtherprovided herein are methods wherein the method further comprises anoxidation step. Further provided herein are methods wherein the methodfurther comprises a deblocking step, wherein the deblocking stepcomprises applying an electrical potential to the at least one sidewallelectrode to generate a deprotection reagent.

Provided herein are methods for storing information, comprising: (a)providing a solid support comprising a surface; (b) depositing at leastone nucleoside on the surface, wherein the at least one nucleosidecouples to a polynucleotide attached to the surface; and (c) repeatingstep b) to synthesize a plurality of polynucleotides on the surface,wherein polynucleotides having different sequences on the surface arepresent at a density of at least 100×10⁶ polynucleotides per cm².Further provided herein are methods wherein the density of addressableloci on the solid support is at least 100×10⁷ polynucleotides per cm².Further provided herein are methods wherein the density of addressableloci on the solid support is 100×10⁶ to 100×10⁷ polynucleotides per cm².Further provided herein are methods wherein the method further comprisescleaving at least one polynucleotide from the surface, wherein thepolynucleotide is dissolved in a droplet. Further provided herein aremethods wherein the method further comprises sequencing at least onepolynucleotide from the surface. Further provided herein are methodswherein the nucleoside comprises a nucleoside phosphoramidite. Furtherprovided herein are methods wherein the method further comprises dryingthe surface. Further provided herein are methods wherein the methodfurther comprises washing the nucleosides away from the surface. Furtherprovided herein are methods wherein the method further comprises acapping step. Further provided herein are methods wherein the methodfurther comprises an oxidation step. Further provided herein are methodswherein the method further comprises a deblocking step.

Provided herein are methods for storing information, comprising: (a)providing a solid support comprising a surface; (b) depositing atdroplet comprising at least one nucleoside on the surface, wherein theat least one nucleoside couples to a polynucleotide attached to thesurface; and (c) repeating step b) to synthesize a plurality ofpolynucleotides on the surface, wherein the droplet has a volume of lessthan about 100 femtoliters. Further provided herein are methods whereinthe droplet has a volume of less than about 50 femtoliters. Furtherprovided herein are methods wherein the droplet has a volume of lessthan about 25 femtoliters to 100 femtoliters. Further provided hereinare methods wherein the method further comprises cleaving at least onepolynucleotide from the surface, wherein the polynucleotide is dissolvedin a droplet. Further provided herein are methods wherein the methodfurther comprises sequencing at least one polynucleotide from thesurface. Further provided herein are methods wherein the nucleosidecomprises a nucleoside phosphoramidite. Further provided herein aremethods wherein the method further comprises drying the surface. Furtherprovided herein are methods wherein the method further comprises washingthe nucleosides away from the surface. Further provided herein aremethods wherein the method further comprises a capping step. Furtherprovided herein are methods wherein the method further comprises anoxidation step. Further provided herein are methods wherein the methodfurther comprises a deblocking step.

Provided herein are methods for storing information, comprising: (a)providing a solid support comprising a surface; (b) depositing at leastone nucleoside on the surface, wherein the at least one nucleosidecouples to a polynucleotide attached to the surface; and (c) repeatingstep b) to synthesize a plurality of polynucleotides on the surface,wherein the time to repeat step b) using four different nucleotides isless than about 100 milliseconds. Further provided herein are methodswherein the time to repeat step b) using four different nucleotides isless than about 50 milliseconds. Further provided herein are methodswherein the time to repeat step b) using four different nucleotides is25 milliseconds to 100 milliseconds. Further provided herein are methodswherein the method further comprises cleaving at least onepolynucleotide from the surface, wherein the polynucleotide is dissolvedin a droplet. Further provided herein are methods further comprisingsequencing at least one polynucleotide from the surface. Furtherprovided herein are methods wherein the nucleoside comprises anucleoside phosphoramidite. Further provided herein are methods whereinthe method further comprises drying the surface. Further provided hereinare methods wherein the method further comprises washing the nucleosidesaway from the surface. Further provided herein are methods wherein themethod further comprises a capping step. Further provided herein aremethods wherein the method further comprises an oxidation step. Furtherprovided herein are methods wherein the method further comprises adeblocking step.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an exemplary workflow for nucleic acid-based datastorage.

FIG. 2 illustrates a plate configured for polynucleotide synthesiscomprising 24 regions, or sub-fields, each having an array of 256clusters.

FIG. 3 illustrates a closer view of the sub-field in FIG. 2 having 16×16of clusters, each cluster having 121 individual loci.

FIG. 4 illustrates a detailed view of the cluster in FIG. 2, where thecluster has 121 loci.

FIG. 5A illustrates a front view of a plate with a plurality ofchannels.

FIG. 5B illustrates a sectional view of plate with a plurality ofchannels.

FIGS. 6A-6B depict a continuous loop and reel-to-reel arrangements forflexible structures.

FIGS. 6C-6D depict schemas for release and extraction of synthesizedpolynucleotides.

FIGS. 7A-7C depict a zoom in of a flexible structure, having spots,channels, or wells, respectively.

FIG. 8 illustrates an example of a computer system.

FIG. 9 is a block diagram illustrating architecture of a computersystem.

FIG. 10 is a diagram demonstrating a network configured to incorporate aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 11 is a block diagram of a multiprocessor computer system using ashared virtual address memory space.

FIG. 12A is a front side of an example of a solid support array.

FIG. 12B is a back side of an example of a solid support array.

FIG. 13 is a schema of solid support comprising an active area andfluidics interface.

FIG. 14 is an example of rack-style instrument.

FIG. 15 depicts a solid support comprising addressable regions fornucleic acid synthesis or storage.

FIG. 16A depicts an array for synthesis using electrochemistry.

FIG. 16B depicts an array for synthesis using electrochemistry.

FIG. 17 depicts wells for nucleic acid synthesis or storage and a pitchdistance between wells.

FIG. 18 illustrates an example of a solid support comprising anaddressable array.

FIG. 19 illustrates an example of a solid support array, wherein thepitch approximates the length of a 240 mer polynucleotide.

DETAILED DESCRIPTION OF THE INVENTION

There is a need for larger capacity storage systems as the amount ofinformation generated and stored is increasing exponentially.Traditional storage media have a limited capacity and requirespecialized technology that changes with time, requiring constanttransfer of data to new media, often at a great expense. A biomoleculesuch as a DNA molecule provides a suitable host for information storagein-part due to its stability over time and capacity for four bitinformation coding, as opposed to traditional binary information coding.Thus, large amounts of data are encoded in the DNA in a relativelysmaller amount of physical space than used by commercially availableinformation storage devices. Provided herein are methods to increase DNAsynthesis throughput through increased sequence density and decreasedturn-around time.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong.

Throughout this disclosure, numerical features are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

As used herein, the terms “preselected sequence”, “predefined sequence”or “predetermined sequence” are used interchangeably. The terms meanthat the sequence of the polymer is known and chosen before synthesis orassembly of the polymer. In particular, various aspects of the inventionare described herein primarily with regard to the preparation of nucleicacids molecules, the sequence of the polynucleotide being known andchosen before the synthesis or assembly of the nucleic acid molecules.

Provided herein are methods and compositions for production of synthetic(i.e. de novo synthesized or chemically synthesized) polynucleotides.Polynucleotides may also be referred to as oligonucleotides or oligos.Polynucleotide sequences described herein may be, unless statedotherwise, comprise DNA or RNA.

Solid Support Based Nucleic Acid Synthesis and Storage

Described herein are devices, compositions, systems and methods for chipbased nucleic acid synthesis and storage. In some instances,polynucleotides are de novo synthesized using solid support basedmethods as described herein. In some instances, polynucleotides arestored on a solid support following synthesis. In some instances, solidsupport based methods as described herein are used for storage only.

Described herein are devices, compositions, systems and methods forsolid support based nucleic acid synthesis and storage, wherein one ormore nucleic acid synthesizer components are integrated into a solidsupport. Components or functional equivalents of components may comprisetemperature control units, addressable electrodes, semiconductingsurfaces, fluid reservoirs, fluidics, synthesis surfaces, power sources,or other component used to synthesize polynucleotides. Any combinationof integrated components is suitable for use with the devices,compositions, systems and methods described herein. In some instances,one or more components is external (non-integrated) to the solidsupport.

The density of unique loci for polynucleotide synthesis on a surface isoften controlled by the spatial resolution achievable by reagentdeposition. Additionally, reagent deposition at unique sites requiresmovement of either the deposition device or the receiving surface tomove the site of reagent deposition from one site to another.Alternatively, coupling of bases is locally controlled at defined siteson a synthesis surface without movement of the surface or a reagentdeposition device. Local control is achieved through an array ofaddressable electrodes 1503, wherein each electrode controls nucleoside(nucleoside phosphoramidite) coupling through electrochemistry at aspecific loci on the surface 1505. In some instances, an electrode is abase electrode (or bottom electrode), located at a bottom region inaddressable communication with the synthesis surface. In some instances,electrodes are sidewall electrodes, located in the side of a well.However, the conventional process of electrochemistry on a flat array insome instances is limited by the pitch distance (See FIG. 19). At highdensities (e.g., short pitch distance 1507), the length of growing DNAoligos 1601 can reach from one synthesis site to the adjacent ones,mixing discrete reaction products. To avoid mixing of adjacent reactionproducts, the pitch distance 1507 in some embodiments is increased.Diffusion through the liquid reaction medium 1901 is an additionalfactor which influences spatial control of polynucleotide synthesis.Thus, it is advantageous to isolate active sites to achieve higher arraydensities. Nucleoside coupling is controlled by local electrodes throughany number of manipulations such as generation of local chemicalreagents, local removal of reagents, repulsion of reagents, restrictionof solvent, attraction of solvent, or other electrochemical or physicalmanipulation that influences one or more steps in base coupling. In asome instances, a device 1500 comprising a plate 1501 with wells 1502 isused to synthesize polynucleotides (See FIG. 15). The bottom of eachwell 1505 comprises an electrode 1503 from which polynucleotides aresynthesized. Each well has a cross-sectional diameter 1506, and a pitchdistance between any two wells 1507. The sidewalls 1504 of the electrodein some instances comprise one or more sidewall electrodes (not shown).

Coupling in some instances is controlled directly by control of thenucleoside addition step, a deprotection step, or other step thataffects the efficiency of a nucleoside coupling reaction. In someinstances, a pattern of electrodes are charged to generate a gradient ofH⁺ ions 1602 on defined sites near the synthesis surface (See FIG. 16A);the polynucleotides 1601 at these sites are unblocked (wherein thepolynucleotide is blocked with an acid-cleavable blocking group) andwill be available for coupling to nucleosides.

Described herein are devices comprising a solid support, wherein thesolid support comprises a plurality of wells, wherein each of the wellscomprises an addressable locus comprising: a synthesis surface locatedin a bottom region of each of the wells; and at least one sidewallelectrode located on a sidewall of each of the wells, wherein theelectrochemical generation of reagents is spatially separated from apolynucleotide attachment point to the synthesis surface. In someinstances, devices described herein further comprise a bottom electrodein addressable communication with the synthesis surface. For example,sidewall electrodes 1603 can be used to control adhesion of substratesor reagents (See FIG. 16B). In some instances, reagents comprise protonsor other acid molecule. In some instances, sidewall electrodes 1603 arelocated at positions around the edges of the well surface (See FIG. 16B)of a well having depth 1604. In some instances, sidewall electrodescontrol chemical reactions occurring near the synthesis surface. Forexample, if acid or other reagent is generated near the synthesissurface, the portion of a polynucleotide 1601 bound to this surface willbe contacted with a higher concentration of acid than the portion of thepolynucleotide that is distal to the site of acid generation. This maylead to degradation of the portion of the polynucleotide which isexposed to higher concentrations of acid. Sidewall electrodes 1603 insome instances produce or control a proton gradient 1602 which resultsin uniform or targeted exposure of a portion of the polynucleotide 1601to acid. Sites near uncharged electrodes do not couple with nucleosidesdeposited over the synthesis surface, and the pattern of chargedelectrodes is altered before addition of the next nucleoside. Byapplying a series of electrode-controlled masks to the surface, thedesired polynucleotides are synthesized at exact locations on thesurface. Additionally, local control of coupling in some instancesreduces synthetic steps, reduces reagents/materials (due to higherpolynucleotide density and reduced scale), and reduces synthesis time(no movement of the synthesis surface). Wells in some instances compriseone, two, three, four, or more than four sidewall electrodes. In someinstances, wells comprise two sidewall electrodes. In some instances,each sidewall electrode is independently addressable. For example,different voltages are independently applied to two or more differentsidewall electrodes. Such arrangements in some instances facilitatediffusion of reagents or polynucleotides in a defined plane between thetwo sidewall electrodes. Such sidewall electrodes in some instances arering-shaped or continuous around the circumference of the wellcross-section. In some instances, sidewall electrodes are discontinuous,or only partially cover a portion of a sidewall surface. For example, asidewall electrode is continuous over about 5%, 10% 15%, 30%, 50%, 75%,or about 90% of the circumference of the well cross-section. Suchsidewall electrodes in some instances have a height about equal to thewell height, or about 5%, 10% 15%, 30%, 50%, 75%, or about 90% of thewell height. In some instances, application of different voltagesindependently to two or more discontinuous sidewall electrodes causesdiffusion of reagents or polynucleotides in a horizontal plane. In someinstances, application of different voltages independently to two ormore discontinuous sidewall electrodes causes diffusion of reagents orpolynucleotides in a vertical plane.

Polynucleotide synthesis generally requires repeated deposition andremoval of liquids (fluidics) on the synthesis surface. Bulk movement offluids is some instances results in fluid loss (wetting, volume oftransport lines or reaction wells), which results in low efficiency ofreagent usage and higher cycle times for moving fluids. An alternativeto bulk fluidics during synthesis is the use of digital fluidics,wherein reagents or reaction vessels are packaged as discrete droplets.Droplets in some instances are mixed, moved (merged, reacted), split,stored, added, removed or analyzed in discrete volumes by manipulationthrough a surface comprising insulated (or semiconductor-coated)electrodes, or electrowetting. Electrowetting allows for local controlof fluid-surface interaction; for example energizing an electrode near adroplet results in splitting of the droplet. In some instances, dropletsas described herein comprise a small volume. For example, the volume ofa droplet is up to 10, 20, 50, 75, 100, 125, 150, 200, 300, 500, 800, ormore than 1000 femtoliters. In some instances, the volume of a dropletis about 50 to about 200 femtoliters. In some instances, digitalfluidics results in at least a 2, 3, 4, 7, 10 or more than 10× decreasein cycle times relative to bulk fluidics. In some instances, digitalfluidics results in about 2× to 10× decrease in cycle times relative tobulk fluidics. In some instances, the time to complete one cycle(sequential coupling of 4 bases, including washes) is about 1, 2, 3, 5,7, 10, 12, 15, 17, 20, 30, 50, 100, or about 200 milliseconds (ms). Insome instances, the time to complete one cycle is up to 1, 2, 3, 5, 7,10, 12, 15, 17, 20, 30, 50, 100, or up to 200 ms. In some instances, thetime to complete one cycle is about 10 to about 50 ms.

Movement of fluids in or out of surfaces described herein may comprisemodifications or conditions that prevent unwanted fluid movement orother phenomenon. For example, fluid movement in some instances resultsin the formation of bubbles or pockets of gas, which limits contact offluids with components such as surfaces or polynucleotides. Variousmethods to control or minimize bubble formation are contemplated by themethods, systems, and compositions described herein. Such methodsinclude control of fluid pressure, well geometry, or surfacematerials/coatings. Well geometry can be implemented to minimizebubbles. For example, tapering the well, channels, or other surface canreduce or eliminate bubble formation during fluid flow. Surfacematerials possessing specific wetting properties can be implemented toreduce or eliminate bubble formation. For example, surfaces describedherein comprise hydrophobic materials. In some instances, surfacesdescribed herein comprise hydrophilic materials. Pressure can be used tocontrol bubble formation during fluid movement. Pressure in someinstances is applied locally to a component, an area of a surface, acapillary/channel, or applied to an entire system. Pressure is in someinstances applied either behind the direction of fluid movement, or infront of it. In some instances, back pressure is applied to prevent theformation of bubbles. Suitable pressures used for preventing bubbleformation can range depending on fluid, the scale, flow geometry, andthe materials used. For example, 5 to 10 atmospheres of pressure aremaintained in the system. In some instances, at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 15, 20 or more than 50 atmospheres of pressure areapplied. In some instances, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,15, 20 or up to 50 atmospheres of pressure are applied. In someinstances, about 2 to about 10, about 2 to about 8, about 2 to about 5,about 4 to about 10, about 4 to about 12, about 5 to about 15, about 5to about 7, about 7 to about 20, about 8 to about 15, or about 10 toabout 20 atmospheres of pressure are applied.

Devices described herein may utilize control units for the purpose ofregulating environmental conditions, such as temperature. Temperaturecontrol units are often used to prepare or maintain conditions forstoring solid supports comprising polynucleotides. Storage conditions ofnucleic acids can affect their long term stability, which directlyinfluences the quality of the digital storage information that isretrieved. Polynucleotides are optionally stored at low temperature (forexample, 10 degrees C., 4 degrees C., 0 degrees C., or lower) on a solidsupport, wherein a temperature control unit maintains this solid supporttemperature. The storage medium for polynucleotides on a solid support,such as solvated or dry also influences storage stability. In someinstances, polynucleotides are stored in solution, such as an aqueoussolution or buffer in droplets. In some instances, polynucleotides arestored lyophilized (dry). Temperature control units in some instancesincrease the chip temperature to facilitate drying of polynucleotidesattached thereto. Temperature control units also provide for localcontrol of heating at addressable locations on the solid support in someinstances. In some instances, following addition of the dropletscomprising the polynucleotides to the solid support, the solid supportis dried. In some instances, the dried solid support is laterresolvated. In some instances, the solid support is stored for lateruse. In some instances, the solid support further comprises an index mapof the polynucleotides. In some instances, the solid support furthercomprises metadata.

Devices described herein can comprise power sources used to energizevarious components of the device. Synthesis components in the solidsupport are optionally powered by an external power source, or a powersource integrated into the solid support. Power sources may comprisebatteries, solar cells, thermoelectric generators, inductive (wireless)power units, kinetic energy charger, cellular telephones, tablets, orother power source suitable for use with the synthesis components ordevices described herein. In some instances, synthesis components,surfaces, or devices described herein are portable.

Fluids comprising reagents, wash solvents, or other synthesis componentsare deposited on the synthesis surface. Unused fluid (prior to contactwith the synthesis surface) or waste fluid (after contact with thesynthesis surface) is in some instances stored in one or morecompartments integrated into the solid support. Alternately or incombination, polynucleotides are moved in or out of the solid supportfor external analysis or storage. For example, synthesizedpolynucleotides are cleaved from loci on the solid support in a droplet,the resulting droplet moved externally to the synthesis area of thesolid support. The droplet is optionally dried for storage. In someinstances, fluids are stored externally from the solid support. In someinstances, a device described herein comprises a solid support with aplurality of fluidics ports which allow movement of fluids in and out ofthe solid support. In some instances, ports are oriented on the sides ofthe solid support, by other configurations are also suitable fordelivery of fluids to the synthesis surface. Such a device oftencomprises, for example, at least 1, 2, 5, 10, 20, 50, 100, 200, 500,1000, 2000, 5000, or at least 10,000 ports per mm length of a solidsupport. In some instances, a device described herein comprises about100 to about 5000 ports per mm per length of a solid support.

Described herein are addressable electrodes integrated into a solidsupport. Electrodes comprise without limitation conductors, insulators,or semi-conductors, and are fabricated of materials well known in theart. Materials may comprise metals, non-metals, mixed-metal oxides,nitrides, carbides, silicon-based materials, or other material. In someinstances, metal oxides include TiO₂, Ta₂O₅, Nb₂O₅, Al₂O₃, BaO, Y₂O₃,HfO₂, SrO or other metal oxide known in the art. In some instances,metal carbides include TiC, WC, ThC₂, ThC, VC, W₂C, ZrC, HfC, NbC, TaC,Ta₂C, or other metal carbide known in the art. In some instances, metalnitrides include GaN, InN, BN, Be₃N₂, Cr₂N, MoN, Si₃N₄, TaN, Th₂N₂, VN,ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitride known in the art. Insome instances, a device disclosed herein is manufactured with acombination of materials listed herein or any other suitable materialknown in the art. Electrodes can possess any shape, including discs,rods, wells, posts, a substantially planar shape, or any other formsuited for nucleic acid synthesis. The or cross-sectional area of eachelectrode varies as a function of the size of the loci forpolynucleotide synthesis, but in some instances is up to 500 um², 200um², 100 um², 75 um², 50 um², 25 um², 10 um², less than 5 um². In someinstances, the cross-sectional area of each electrode is about 500 um²to 10 um², about 100 um² to 25 um², or about 150 um² to 50 um². In someinstances, the cross-sectional area of each electrode is about 150 um²to 50 um². Devices provide herein include electrodes having a diameterthat varies as a function of the size of the loci for polynucleotidesynthesis. Exemplary electrode diameters include, without limitation, upto 500 um, 200 um, 100 um, 75 um, 50 um, 25 um, 10 um, less than 5 um.In some instances, the diameter of each electrode is about 500 um to 10um, about 100 um to 25 um, about 100 um to about 200 um, about 50 um toabout 200 um, or about 150 um to 50 um. In some instances, the diameterof each electrode is about 200 um to 50 um. In some instances, thediameter of each electrode is about 200 um to 100 um. In some instances,the diameter of each electrode is up to 500 nm, 200 nm, 100 nm, 75 nm,50 nm, 25 nm, 10 nm, less than 5 nm. In some instances, the diameter ofeach electrode is about 500 nm to 10 nm, about 100 nm to 25 nm, about100 nm to about 200 nm, about 50 nm to about 200 nm, or about 150 nm to50 nm. In some instances, the diameter of each electrode is about 200 nmto 50 nm. In some instances, the diameter of each electrode is about 200nm to 100 nm. The thickness of each electrode varies as a function ofthe size of the loci for polynucleotide synthesis, but in some instancesis about 50 nm, 100 nm, 200 nm, 500 nm, 750 nm, 1000 nm, 1200 nm, 1500nm, 2000 nm, 2500 nm, 3000 nm, or about 3500 nm. In some instances thethickness of the electrode is at least 50 nm, 100 nm, 200 nm, 500 nm,750 nm, 1000 nm, 1200 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, or atleast 3500 nm. In some instances the thickness of the electrode is atleast 1 um, 2 um, 3 um, 5 um, 10 um, 15 um, 20 um, 30 um, 50 um or atleast 75 um. In some instances the thickness of the electrode is about 1um, 2 um, 3 um, 5 um, 10 um, 15 um, 20 um, 30 um, 50 um or about 75 um.In some instances the thickness of the electrode is up to 1 um, 2 um, 3um, 5 um, 10 um, 15 um, 20 um, 30 um, 50 um or up to 75 um. In someinstances the thickness of the electrode is up to 50 nm, 100 nm, 200 nm,500 nm, 750 nm, 1000 nm, 1200 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, orup to 3500 nm. In some instances the thickness of the electrode is about20 nm to 3000 nm, about 50 nm to 2500, about 100 nm to 750 nm, about 400nm to 750 nm, about 500 nm to 3000 nm, or about 1000 nm to 3000 nm. Insome instances the thickness of the electrode is about 10 um to about 20um. In some instances the thickness of the electrode is about 5 um toabout 50 um, about 10 um to about 30 um, about 15 um to about 25 um, orabout 30 um to about 50 um. In some instances, electrodes are coatedwith additional materials such as semiconductors or insulators. In someinstances, electrodes are coated with materials for polynucleotideattachment and synthesis. The size, shape, pattern, or orientation ofelectrodes is in some instances chosen to minimize deleterious sidereactions caused by electrochemically generated reagents. In someinstances combinations of electrodes are used, such as a grid ofaddressable electrodes and a common electrode. Electrodes are in someinstances cathodes or anodes. Electrodes or arrays of electrodes can bepositioned anywhere on or near the polynucleotide surface. In someinstances, electrodes are placed on the bottom of loci for synthesis,such as the bottom of a well or channel. In some instances, electrodesare placed in the sidewalls of a well or channel (“sidewallelectrodes”). A plurality of sidewall electrodes are in some instancespresent on the sides of a well. Electrodes positioned at differentlocations in the device can have different functions, and areindependently or simultaneously addressable. In some instances, anelectrode in the bottom of a well is used to cleave polynucleotides fromthe surface at one or more loci, and a sidewall electrode is used togenerate acid to deprotect polynucleotides. In some instances, anelectrode in the bottom of a well is used to cleave polynucleotides fromthe surface at one or more loci, and a first sidewall electrode is usedto generate acid to deprotect polynucleotides, and a second sidewallelectrode is used to move polynucleotides out of the well aftercleavage. In exemplary configurations, sidewall electrodes are locatedabout 10 nm, about 25 nm, about 50 nm, about 75 nm, about 100 nm, about125 nm, or about 200 nm above the synthesis surface. In some instances,sidewall electrodes are located about 10 nm to about 100 nm, about 50 nmto about 150 nm, about 40 nm to 100 nm, about 75 nm to about 125 nm,about 100 to 300 nm above the synthesis surface. In some instances,multiple sidewall electrodes are located at different heights above thesynthesis surface. For example, a locus comprises at least one sidewallelectrode, at least 2 sidewall electrodes, at least 3 sidewallelectrodes, or more than 3 sidewall electrodes. In exemplaryconfigurations, sidewall electrodes have a height of about 1 nm, about 5nm, about 10 nm, about 20 nm, about 25 nm about 30 nm, about 40 nm, orabout 50 nm. In some instances, sidewall electrodes have a height of 1nm to 20 nm, 2 nm to 30 nm, 5 nm to 20 nm, 10 nm to 40 nm, or 5 nm to 25nm.

Electrode surfaces can support the movement, conformation, synthesis,growth, and release of polynucleotides. In some instances, electrodesare coated with one or more layers. In some instances, the layer is amonolayer which facilities attachment of a linker. In some instances,the electrode is charged to influence the area of the monolayer to befunctionalized with a linker. This allows for masking of specific areasfor chemical functionalization, such as modifying the surface withhydrophobic or hydrophilic chemical groups. In some instances, theelectrode is charged to influence the area of the monolayer to beextended with a nucleoside monomer. This in some instances includesgeneration of reagents to facilitate or prevent coupling of monomers tosynthesis surfaces in the vicinity of an electrode. In some instances,the electrode is charged to influence the area of the monolayer whichreleases polynucleotides. Such controlled release of specificpolynucleotides in a specific order in some instances is used to controlthe assembly of synthesized monomers into larger polynucleotides. Forexample, an iterative polynucleotide assembly process is optimized byexploring which various combinations of polynucleotides that arereleased and allowed to hybridize for overlap PCR assembly.

Each electrode can control one, or a plurality of different loci forsynthesis, wherein each locus for synthesis has a density ofpolynucleotides. In some instances, the density is at least 1 oligo per10 nm², 20, 50, 100, 200, 500, 1,000, 2,000, 5,000 or at least 1 oligoper 10,000 nm². In some instances, the density is about 1 oligo per 10nm² to about 1 oligo per 5,000 nm², about 1 oligo per 50 nm² to about 1oligo per 500 nm², or about 1 oligo per 25 nm² to about 1 oligo per 75nm². In some instances, the density of polynucleotides is about 1 oligoper 25 nm² to about 1 oligo per 75 nm².

Provided herein are devices for polynucleotide synthesis having varioustypes of electrodes. In some instances, the device comprises a referenceelectrode. Reference electrodes are placed near the synthesis surface orin the case of a well or channel, for example, above a well or channel.In some instances, a reference electrode is about 1 to about 50 um abovethe synthesis surface, about 2 um to about 40 um, about 3 um to about 30um, about 5 um to about 20 um, about 10 to about 20 um, about 15 toabout 50 um, about 30 to about 50 um, about 5 um to about 30 um or about7 um to about 25 um. In some instances a reference electrode is about 1,2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 um or more than 26 umabove the synthesis surface. In some instances a reference electrode isup to 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 um or up to26 um above the synthesis surface. In some instances a referenceelectrode is at least 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24,26 um or at least 26 um above the synthesis surface. Referenceelectrodes in some instances are adjacent to synthesis surfaces, such asadjacent to a well or channel. Each locus in some instances has onecorresponding reference electrode. In some instances, each locus sharesa common reference electrode with one or more adjacent loci. The devicesdescribed herein may comprise any number of reference electrodes. Insome instances the reference electrode is a single, uniform plate. Insome instances, devices comprise a plurality of reference electrodes. Areference electrode can address a plurality of loci, for example 2, 3,4, 5, 10 or more loci.

Described herein are devices, compositions, systems and methods forsolid support based nucleic acid synthesis and storage, wherein thesolid support has varying dimensions. In some instances, a size of thesolid support is between about 40 and 120 mm by between about 25 and 100mm. In some instances, a size of the solid support is about 80 mm byabout 50 mm. In some instances, a width of a solid support is at leastor about 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, 100 mm, 150 mm, 200 mm, 300mm, 400 mm, 500 mm, or more than 500 mm. In some instances, a height ofa solid support is at least or about 10 mm, 20 mm, 40 mm, 60 mm, 80 mm,100 mm, 150 mm, 200 mm, 300 mm, 400 mm, 500 mm, or more than 500 mm. Insome instances, the solid support has a planar surface area of at leastor about 100 mm²; 200 mm²; 500 mm²; 1,000 mm²; 2,000 mm²; 4,500 mm²;5,000 mm²; 10,000 mm²; 12,000 mm²; 15,000 mm²; 20,000 mm²; 30,000 mm²;40,000 mm²; 50,000 mm² or more. In some instances, the thickness of thesolid support is between about 50 mm and about 2000 mm, between about 50mm and about 1000 mm, between about 100 mm and about 1000 mm, betweenabout 200 mm and about 1000 mm, or between about 250 mm and about 1000mm. Non-limiting examples thickness of the solid support include 275 mm,375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In someinstances, the thickness of the solid support is at least or about 0.5mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than4.0 mm.

Described herein are devices wherein two or more solid supports areassembled. In some instances, solid supports are interfaced together ona larger unit. Interfacing may comprise exchange of fluids, electricalsignals, or other medium of exchange between solid supports. This unitis capable of interface with any number of servers, computers, ornetworked devices. For example, a plurality of solid support isintegrated onto a rack unit, which is conveniently inserted or removedfrom a server rack. The rack unit may comprise any number of solidsupports. In some instances the rack unit comprises at least 1, 2, 5,10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000,100,000 or more than 100,000 solid supports. In some instances, two ormore solid supports are not interfaced with each other. Nucleic acids(and the information stored in them) present on solid supports can beaccessed from the rack unit. See e.g., FIG. 14. Access includes removalof polynucleotides from solid supports, direct analysis ofpolynucleotides on the solid support, or any other method which allowsthe information stored in the nucleic acids to be manipulated oridentified. Information in some instances is accessed from a pluralityof racks, a single rack, a single solid support in a rack, a portion ofthe solid support, or a single locus on a solid support. In variousinstances, access comprises interfacing nucleic acids with additionaldevices such as mass spectrometers, HPLC, sequencing instruments, PCRthermocyclers, or other device for manipulating nucleic acids. Access tonucleic acid information in some instances is achieved by cleavage ofpolynucleotides from all or a portion of a solid support. Cleavage insome instances comprises exposure to chemical reagents (ammonia or otherreagent), electrical potential, radiation, heat, light, acoustics, orother form of energy capable of manipulating chemical bonds. In someinstances, cleavage occurs by charging one or more electrodes in thevicinity of the polynucleotides. In some instances, electromagneticradiation in the form of UV light is used for cleavage ofpolynucleotides. In some instances, a lamp is used for cleavage ofpolynucleotides, and a mask mediates exposure locations of the UV lightto the surface. In some instances, a laser is used for cleavage ofpolynucleotides, and a shutter opened/closed state controls exposure ofthe UV light to the surface. In some instances, access to nucleic acidinformation (including removal/addition of racks, solid supports,reagents, nucleic acids, or other component) is completely automated.

Solid supports as described herein comprise an active area. In someinstances, the active area comprises addressable regions or loci fornucleic acid synthesis. In some instances, the active area comprisesaddressable regions or loci for nucleic acid storage.

The active area comprises varying dimensions. For example, the dimensionof the active area is between about 1 mm to about 50 mm by about 1 mm toabout 50 mm. In some instances, the active area comprises a width of atleast or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm. In some instances,the active area comprises a height of at least or about 0.5, 1, 1.5, 2,2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, or more than 80 mm. An exemplary active area within a solid supportis seen in FIG. 13. A package 1307 comprises an active area 1305 withina solid support 1303. The package 1307 also comprises a fluidicsinterface 1301.

Described herein are devices, compositions, systems and methods forsolid support based nucleic acid synthesis and storage, wherein thesolid support has a number of sites (e.g., spots) or positions forsynthesis or storage. In some instances, the solid support comprises upto or about 10,000 by 10,000 positions in an area. In some instances,the solid support comprises between about 1000 and 20,000 by betweenabout 1000 and 20,000 positions in an area. In some instances, the solidsupport comprises at least or about 10, 30, 50, 75, 100, 200, 300, 400,500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,12,000, 14,000, 16,000, 18,000, 20,000 positions by least or about 10,30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000positions in an area. In some instances the area is up to 0.25, 0.5,0.75, 1.0, 1.25, 1.5, or 2.0 inches squared. In some instances, thesolid support comprises addressable loci having a pitch of at least orabout 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5, 6, 7, 8, 9, 10, or more than 10 um. In some instances, the solidsupport comprises addressable loci having a pitch of about 5 um. In someinstances, the solid support comprises addressable loci having a pitchof about 2 um. In some instances, the solid support comprisesaddressable loci having a pitch of about 1 um. In some instances, thesolid support comprises addressable loci having a pitch of about 0.2 um.In some instances, the solid support comprises addressable loci having apitch of about 0.2 um to about 10 um, about 0.2 to about 8 um, about 0.5to about 10 um, about 1 um to about 10 um, about 2 um to about 8 um,about 3 um to about 5 um, about 1 um to about 3 um or about 0.5 um toabout 3 um. In some instances, the solid support comprises addressableloci having a pitch of about 0.1 um to about 3 um. See e.g. FIG. 15,FIG. 16A, and FIG. 16B.

The solid support for nucleic acid synthesis or storage as describedherein comprises a high capacity for storage of data. For example, thecapacity of the solid support is at least or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, ormore than 1000 petabytes. In some instances, the capacity of the solidsupport is between about 1 to about 10 petabytes or between about 1 toabout 100 petabytes. In some instances, the capacity of the solidsupport is about 100 petabytes. In some instances, the data is stored asaddressable arrays of packets as droplets. In some instances, the datais stored as addressable arrays of packets as droplets on a spot. Insome instances, the data is stored as addressable arrays of packets asdry wells. In some instances, the addressable arrays comprise at leastor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than200 gigabytes of data. In some instances, the addressable arrayscomprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100,200, or more than 200 terabytes of data. In some instances, an item ofinformation is stored in a background of data. For example, an item ofinformation encodes for about 10 to about 100 megabytes of data and isstored in 1 petabyte of background data. In some instances, an item ofinformation encodes for at least or about 1, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 300, 400, 500, or more than 500 megabytes of dataand is stored in 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,300, 400, 500, or more than 500 petabytes of background data.

Provided herein are devices, compositions, systems and methods for solidsupport based nucleic acid synthesis and storage, wherein followingsynthesis, the polynucleotides are collected in packets as one or moredroplets. In some instances, the polynucleotides are collected inpackets as one or more droplets and stored. In some instances, a numberof droplets is at least or about 1, 10, 20, 50, 100, 200, 300, 500,1000, 2500, 5000, 75000, 10,000, 25,000, 50,000, 75,000, 100,000, 1million, 5 million, 10 million, 25 million, 50 million, 75 million, 100million, 250 million, 500 million, 750 million, or more than 750 milliondroplets. In some instances, a droplet volume comprises 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or morethan 100 um (micrometer) in diameter. In some instances, a dropletvolume comprises 1-100 um, 10-90 um, 20-80 um, 30-70 um, or 40-50 um indiameter.

In some instances, the polynucleotides that are collected in the packetscomprise a similar sequence. In some instances, the polynucleotidesfurther comprise a non-identical sequence to be used as a tag orbarcode. For example, the non-identical sequence is used to index thepolynucleotides stored on the solid support and to later search forspecific polynucleotides based on the non-identical sequence. Exemplarytag or barcode lengths include barcode sequences comprising, withoutlimitation, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or morebases in length. In some instances, the tag or barcode comprise at leastor about 10, 50, 75, 100, 200, 300, 400, or more than 400 base pairs inlength.

Provided herein are devices, compositions, systems and methods for solidsupport based nucleic acid synthesis and storage, wherein thepolynucleotides are collected in packets comprising redundancy. Forexample, the packets comprise about 100 to about 1000 copies of eachpolynucleotide. In some instances, the packets comprise at least orabout 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200,1400, 1600, 1800, 2000, or more than 2000 copies of each polynucleotide.In some instances, the packets comprise about 1000× to about 5000×synthesis redundancy. Synthesis redundancy in some instances is at leastor about 500×, 1000×, 1500×, 2000×, 2500×, 3000×, 3500×, 4000×, 5000×,6000×, 7000×, 8000×, or more than 8000×. The polynucleotides that aresynthesized using solid support based methods as described hereincomprise various lengths. In some instances, the polynucleotides aresynthesized and further stored on the solid support. In some instances,the polynucleotide length is in between about 100 to about 1000 bases.In some instances, the polynucleotides comprise at least or about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more than2000 bases in length.

Nucleic Acid Based Information Storage

Provided herein are devices, compositions, systems and methods fornucleic acid-based information (data) storage. An exemplary workflow isprovided in FIG. 1. In a first step, a digital sequence encoding an itemof information (i.e., digital information in a binary code forprocessing by a computer) is received 101. An encryption 103 scheme isapplied to convert the digital sequence from a binary code to a nucleicacid sequence 105. A surface material for nucleic acid extension, adesign for loci for nucleic acid extension (aka, arrangement spots), andreagents for nucleic acid synthesis are selected 107. The surface of astructure is prepared for nucleic acid synthesis 108. De novopolynucleotide synthesis is performed 109. The synthesizedpolynucleotides are stored 111 and available for subsequent release 113,in whole or in part. Once released, the polynucleotides, in whole or inpart, are sequenced 115, subject to decryption 117 to convert nucleicsequence back to digital sequence. The digital sequence is thenassembled 119 to obtain an alignment encoding for the original item ofinformation.

Items of Information

Optionally, an early step of data storage process disclosed hereinincludes obtaining or receiving one or more items of information in theform of an initial code. Items of information include, withoutlimitation, text, audio and visual information. Exemplary sources foritems of information include, without limitation, books, periodicals,electronic databases, medical records, letters, forms, voice recordings,animal recordings, biological profiles, broadcasts, films, short videos,emails, bookkeeping phone logs, internet activity logs, drawings,paintings, prints, photographs, pixelated graphics, and software code.Exemplary biological profile sources for items of information include,without limitation, gene libraries, genomes, gene expression data, andprotein activity data. Exemplary formats for items of informationinclude, without limitation, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls,.xlsx, .rtf, .jpg, .gif, .psd, .bmp, .tiff, .png, and .mpeg. The amountof individual file sizes encoding for an item of information, or aplurality of files encoding for items of information, in digital formatinclude, without limitation, up to 1024 bytes (equal to 1 KB), 1024 KB(equal to 1 MB), 1024 MB (equal to 1 GB), 1024 GB (equal to 1 TB), 1024TB (equal to 1 PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyteor more. In some instances, an amount of digital information is at least1 gigabyte (GB). In some instances, the amount of digital information isat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000 or more than 1000 gigabytes. In some instances,the amount of digital information is at least 1 terabyte (TB). In someinstances, the amount of digital information is at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 or more than 1000 terabytes. In some instances, the amount ofdigital information is at least 1 petabyte (PB). In some instances, theamount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than1000 petabytes.

Structures for Polynucleotide Synthesis

Provided herein are rigid or flexibles structures for polynucleotidesynthesis. In the case of rigid structures, provided herein are deviceshaving a structure for the generation of a library of polynucleotides.In some instances, the structure comprises a plate. An exemplarystructure 200 is illustrated in FIG. 2, wherein the structure 200 hasabout the same size dimensions as a standard 96 well plate: 140 mm by 90mm. The structure 200 comprises clusters grouped in 24 regions orsub-fields 205, each sub-field 205 comprising an array of 256 clusters210. An expanded view of an exemplary sub-field 205 is shown in FIG. 3.In the expanded view of four clusters (FIG. 3), a single cluster 210,has a Y axis cluster pitch (distance from center to center of adjacentclusters) of 1079.210 um or 1142.694 um, and an X axis cluster pitch of1125 um. An illustrative cluster 210 is depicted in FIG. 4, where the Yaxis loci pitch (distance from center to center of adjacent loci) is63.483 um, and an X axis loci pitch is 75 um. The locus width at thelongest part, e.g., diameter for a circular locus, is 50 um and thedistance between loci is 24 um. The number of loci 405 in the exemplarycluster in FIG. 4 is 121. The loci may be flat, wells, or channels. Anexemplary channel arrangement is illustrated in FIGS. 5A-5B where aplate 505 is illustrated comprising a main channel 510 and a pluralityof channels 515 connected to the main channel 510. The connectionbetween the main channel 510 and the plurality of channels 515 providesfor a fluid communication for flow paths from the main channel 510 tothe each of the plurality of channels 515. A plate 505 described hereincan comprise multiple main channels 510. The plurality of channels 515collectively forms a cluster within the main channel 510.

In the case of flexible structures, provided herein are devices whereinthe flexible structure comprises a continuous loop 601 wrapped aroundone or more fixed structures, e.g., a pair of rollers 603 or anon-continuous flexible structure 607 wrapped around separate fixedstructures, e.g., a pair reels 605. See FIGS. 6A-6B. In some instances,the structures comprise multiple regions for polynucleotide synthesis.An exemplary structure is illustrated in FIG. 6C where a plate comprisesdistinct regions 609 for polynucleotide synthesis. The distinct regions609 may be separated 611 by breaking or cutting. Each of the distinctregions may be further released, sequenced, decrypted, and read 613 orstored 615. An alternative structure is illustrated in FIG. 6D in whicha tape comprises distinct regions 617 for polynucleotide synthesis. Thedistinct regions 617 may be separated 619 by breaking or cutting. Eachof the distinct regions may be further released, sequenced, decrypted,and read 621 or stored 623. Provided herein are flexible structureshaving a surface with a plurality of loci for polynucleotide extension.FIGS. 7A-7C show a zoom in of the locus in the flexible structure. Eachlocus in a portion of the flexible structure 701, may be a substantiallyplanar spot 703 (e.g., flat), a channel 705, or a well 707. In someinstances, each locus of the structure has a width of about 10 um and adistance between the center of each structure of about 21 um. Loci maycomprise, without limitation, circular, rectangular, tapered, or roundedshapes. Alternatively or in combination, the structures are rigid. Insome instances, the rigid structures comprise loci for polynucleotidesynthesis. In some instances, the rigid structures comprisesubstantially planar regions, channels, or wells for polynucleotidesynthesis.

In some instances, a well described herein has a width to depth (orheight) ratio of 1 to 0.01, wherein the width is a measurement of thewidth at the narrowest segment of the well. In some instances, a welldescribed herein has a width to depth (or height) ratio of 0.5 to 0.01,wherein the width is a measurement of the width at the narrowest segmentof the well. In some instances, a well described herein has a width todepth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5,or 1. Provided herein are structures for polynucleotide synthesiscomprising a plurality of discrete loci for polynucleotide synthesis.Exemplary structures for the loci include, without limitation,substantially planar regions, channels, wells or protrusions. Structuresdescribed herein are may comprise a plurality of clusters, each clustercomprising a plurality of wells, loci or channels. Alternatively,described herein are may comprise a homogenous arrangement of wells,loci or channels. Structures provided herein may comprise wells having aheight or depth from about 5 um to about 500 um, from about 5 um toabout 400 um, from about 5 um to about 300 um, from about 5 um to about200 um, from about 5 um to about 100 um, from about 5 um to about 50 um,or from about 10 um to about 50 um. In some instances, the height of awell is less than 100 um, less than 80 um, less than 60 um, less than 40um or less than 20 um. In some instances, well height is about 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 um or more. In someinstances, the height or depth of the well is at least 10, 25, 50, 75,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm.In some instances, the height or depth of the well is in a range ofabout 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nmto about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600nm, or about 200 nm to about 500. In some instances, the height or depthof the well is in a range of about 50 nm to about 1 um. In someinstances, well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 700, 800, 900 or about 1000 nm.

Structures for polynucleotide synthesis provided herein may comprisechannels. The channels may have a width to depth (or height) ratio of 1to 0.01, wherein the width is a measurement of the width at thenarrowest segment of the microchannel. In some instances, a channeldescribed herein has a width to depth (or height) ratio of 0.5 to 0.01,wherein the width is a measurement of the width at the narrowest segmentof the microchannel. In some instances, a channel described herein has awidth to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16,0.2, 0.5, or 1.

Described herein are structures for polynucleotide synthesis comprisinga plurality of discrete loci. Structures comprise, without limitation,substantially planar regions, channels, protrusions, or wells forpolynucleotide synthesis. In some instances, structures described hereinare provided comprising a plurality of channels, wherein the height ordepth of the channel is from about 5 um to about 500 um, from about 5 umto about 400 um, from about 5 um to about 300 um, from about 5 um toabout 200 um, from about 5 um to about 100 um, from about 5 um to about50 um, or from about 10 um to about 50 um. In some cases, the height ofa channel is less than 100 um, less than 80 um, less than 60 um, lessthan 40 um or less than 20 um. In some cases, channel height is about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 um or more.In some instances, the height or depth of the channel is at least 10,25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or morethan 1000 nm. In some instances, the height or depth of the channel isin a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm,about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nmto about 600 nm, or about 200 nm to about 500. Channels described hereinmay be arranged on a surface in clusters or as a homogenous field.

The width of a locus on the surface of a structure for polynucleotidesynthesis described herein may be from about 0.1 um to about 500 um,from about 0.5 um to about 500 um, from about 1 um to about 200 um, fromabout 1 um to about 100 um, from about 5 um to about 100 um, or fromabout 0.1 um to about 100 um, for example, about 90 um, 80 um, 70 um, 60um, 50 um, 40 um, 30 um, 20 um, 10 um, 5 um, 1 um or 0.5 um. In someinstances, the width of a locus is less than about 100 um, 90 um, 80 um,70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um. In some instances,the width of a locus is at least 10, 25, 50, 75, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances,the width of a locus is in a range of about 10 nm to about 1000 nm,about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm toabout 700 nm, about 100 nm to about 600 nm, or about 200 nm to about500. In some instances, the width of a locus is in a range of about 50nm to about 1000 nm. In some instances, the distance between the centerof two adjacent loci is from about 0.1 um to about 500 um, 0.5 um toabout 500 um, from about 1 um to about 200 um, from about 1 um to about100 um, from about 5 um to about 200 um, from about 5 um to about 100um, from about 5 um to about 50 um, or from about 5 um to about 30 um,for example, about 20 um. In some instances, the total width of a locusis about 5 um, 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um,90 um, or 100 um. In some instances, the total width of a locus is about1 um to 100 um, 30 um to 100 um, or 50 um to 70 um. In some instances,the distance between the center of two adjacent loci is from about 0.5um to about 2 um, 0.5 um to about 2 um, from about 0.75 um to about 2um, from about 1 um to about 2 um, from about 0.2 um to about 1 um, fromabout 0.5 um to about 1.5 um, from about 0.5 um to about 0.8 um, or fromabout 0.5 um to about 1 um, for example, about 1 um. In some instances,the total width of a locus is about 50 nm, 0.1 um, 0.2 um, 0.3 um, 0.4um, 0.5 um, 0.6 um, 0.7 um, 0.8 um, 0.9 um, 1 um, 1.1 um, 1.2 um, 1.3um, 1.4 um, or 1.5 um. In some instances, the total width of a locus isabout 0.5 um to 2 um, 0.75 um to 1 um, or 0.9 um to 2 um.

In some instances, each locus supports the synthesis of a population ofpolynucleotides having a different sequence than a population ofpolynucleotides grown on another locus. Provided herein are surfaceswhich comprise at least 10, 100, 256, 500, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000,30000, 40000, 50000 or more clusters. Provided herein are surfaces whichcomprise more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 5,000,000; or 10,000,000 or more distinct loci. Insome cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 500 or more loci. Insome cases, each cluster includes 50 to 500, 50 to 200, 50 to 150, or100 to 150 loci. In some cases, each cluster includes 100 to 150 loci.In some instances, each cluster includes 109, 121, 130 or 137 loci.

Provided herein are loci having a width at the longest segment of 5 to100 um. In some cases, the loci have a width at the longest segment ofabout 30, 35, 40, 45, 50, 55 or 60 um. In some cases, the loci arechannels having multiple segments, wherein each segment has a center tocenter distance apart of 5 to 50 um. In some cases, the center to centerdistance apart for each segment is about 5, 10, 15, 20 or 25 um.

In some instances, the number of distinct polynucleotides synthesized onthe surface of a structure described herein is dependent on the numberof distinct loci available in the substrate. In some instances, thedensity of loci within a cluster of a substrate is at least or about 1locus per mm², 10 loci per mm², 25 loci per mm², 50 loci per mm², 65loci per mm², 75 loci per mm², 100 loci per mm², 130 loci per mm², 150loci per mm², 175 loci per mm², 200 loci per mm², 300 loci per mm², 400loci per mm², 500 loci per mm², 1,000 loci per mm², 10⁴ loci per mm²,10⁵ loci per mm², 10⁶ loci per mm², or more. In some cases, a substratecomprises from about 10 loci per mm² to about 500 mm², from about 25loci per mm² to about 400 mm², from about 50 loci per mm² to about 500mm², from about 100 loci per mm² to about 500 mm², from about 150 lociper mm² to about 500 mm², from about 10 loci per mm² to about 250 mm²,from about 50 loci per mm² to about 250 mm², from about 10 loci per mm²to about 200 mm², or from about 50 loci per mm² to about 200 mm². Insome cases, a substrate comprises from about 10⁴ loci per mm² to about10⁵ mm². In some cases, a substrate comprises from about 10⁵ loci permm² to about 10⁷ mm². In some cases, a substrate comprises at least 10⁵loci per mm². In some cases, a substrate comprises at least 10⁶ loci permm². In some cases, a substrate comprises at least 10⁷ loci per mm². Insome cases, a substrate comprises from about 10⁴ loci per mm² to about10⁵ mm². In some instances, the density of loci within a cluster of asubstrate is at least or about 1 locus per um², 10 loci per um², 25 lociper um², 50 loci per um², 65 loci per um², 75 loci per um², 100 loci perum², 130 loci per um², 150 loci per um², 175 loci per um², 200 loci perum², 300 loci per um², 400 loci per um², 500 loci per um², 1,000 lociper um² or more. In some cases, a substrate comprises from about 10 lociper um² to about 500 um², from about 25 loci per um² to about 400 um²,from about 50 loci per um² to about 500 um², from about 100 loci per um²to about 500 um², from about 150 loci per um² to about 500 um², fromabout 10 loci per um² to about 250 um², from about 50 loci per um² toabout 250 um², from about 10 loci per um² to about 200 um², or fromabout 50 loci per um² to about 200 um².

In some instances, the distance between the centers of two adjacent lociwithin a cluster is from about 10 um to about 500 um, from about 10 umto about 200 um, or from about 10 um to about 100 um. In some cases, thedistance between two centers of adjacent loci is greater than about 10um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um or 100 um. Insome cases, the distance between the centers of two adjacent loci isless than about 200 um, 150 um, 100 um, 80 um, 70 um, 60 um, 50 um, 40um, 30 um, 20 um or 10 um. In some cases, the distance between thecenters of two adjacent loci is less than about 10000 nm, 8000 nm, 6000nm, 4000 nm, 2000 nm 1000 nm, 800 nm, 600 nm, 400 nm, 200 nm, 150 nm,100 nm, 80 um, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm or 10 nm. Insome instances, each square meter of a structure described herein allowsfor at least 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ loci, where each locus supportsone polynucleotide. In some instances, 10⁹ polynucleotides are supportedon less than about 6, 5, 4, 3, 2 or 1 m² of a structure describedherein.

In some instances, a structure described herein provides support for thesynthesis of more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000;2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000;5,000,000; 10,000,000 or more non-identical polynucleotides. In somecases, the structure provides support for the synthesis of more than2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000;400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000;1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000;3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 ormore polynucleotides encoding for distinct sequences. In some instances,at least a portion of the polynucleotides have an identical sequence orare configured to be synthesized with an identical sequence. In someinstances, the structure provides a surface environment for the growthof polynucleotides having at least 50, 60, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500 bases or more. In some arrangements,structures for polynucleotide synthesis described herein comprise sitesfor polynucleotide synthesis in a uniform arrangement.

In some instances, polynucleotides are synthesized on distinct loci of astructure, wherein each locus supports the synthesis of a population ofpolynucleotides. In some cases, each locus supports the synthesis of apopulation of polynucleotides having a different sequence than apopulation of polynucleotides grown on another locus. In some instances,the loci of a structure are located within a plurality of clusters. Insome instances, a structure comprises at least 10, 500, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000,14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In someinstances, a structure comprises more than 2,000; 5,000; 10,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000;1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or10,000,000 or more distinct loci. In some cases, each cluster includes1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120,130, 150 or more loci. In some instances, each cluster includes 50 to500, 100 to 150, or 100 to 200 loci. In some instances, each clusterincludes 109, 121, 130 or 137 loci. In some instances, each clusterincludes 5, 6, 7, 8, 9, 10, 11 or 12 loci. In some instances,polynucleotides from distinct loci within one cluster have sequencesthat, when assembled, encode for a contiguous longer polynucleotide of apredetermined sequence.

Structure Size

In some instances, a structure described herein is about the size of aplate (e.g., chip), for example between about 40 and 120 mm by betweenabout 25 and 100 mm. In some instances, a structure described herein hasa diameter less than or equal to about 1000 mm, 500 mm, 450 mm, 400 mm,300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50 mm. In some instances, thediameter of a substrate is between about 25 mm and 1000 mm, betweenabout 25 mm and about 800 mm, between about 25 mm and about 600 mm,between about 25 mm and about 500 mm, between about 25 mm and about 400mm, between about 25 mm and about 300 mm, or between about 25 mm andabout 200. Non-limiting examples of substrate size include about 300 mm,200 mm, 150 mm, 130 mm, 100 mm, 84 mm, 76 mm, 54 mm, 51 mm and 25 mm. Insome instances, a substrate has a planar surface area of at least 100mm²; 200 mm²; 500 mm²; 1,000 mm²; 2,000 mm²; 4,500 mm²; 5,000 mm²;10,000 mm²; 12,000 mm²; 15,000 mm²; 20,000 mm²; 30,000 mm²; 40,000 mm²;50,000 mm² or more. In some instances, the thickness is between about 50mm and about 2000 mm, between about 50 mm and about 1000 mm, betweenabout 100 mm and about 1000 mm, between about 200 mm and about 1000 mm,or between about 250 mm and about 1000 mm. Non-limiting examplesthickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mmand 925 mm. In some instances, the thickness is at least or about 0.5mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than4.0 mm. In some cases, the thickness of varies with diameter and dependson the composition of the substrate. For example, a structure comprisingmaterials other than silicon may have a different thickness than asilicon structure of the same diameter. Structure thickness may bedetermined by the mechanical strength of the material used and thestructure must be thick enough to support its own weight withoutcracking during handling. In some instances, a structure is more thanabout 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any one dimension.

Materials

Provided herein are devices comprising a surface, wherein the surface ismodified to support polynucleotide synthesis at predetermined locationsand with a resulting low error rate, a low dropout rate, a high yield,and a high oligo representation. In some instances, surfaces of devicesfor polynucleotide synthesis provided herein are fabricated from avariety of materials capable of modification to support a de novopolynucleotide synthesis reaction. In some cases, the devices aresufficiently conductive, e.g., are able to form uniform electric fieldsacross all or a portion of the devices. Devices described herein maycomprise a flexible material. Exemplary flexible materials include,without limitation, modified nylon, unmodified nylon, nitrocellulose,and polypropylene. Devices described herein may comprise a rigidmaterial. Exemplary rigid materials include, without limitation, glass,fuse silica, silicon, silicon dioxide, silicon nitride, plastics (forexample, polytetrafluoroethylene, polypropylene, polystyrene,polycarbonate, and blends thereof, and metals (for example, gold,platinum). Devices disclosed herein may be fabricated from a materialcomprising silicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combinationthereof. In some cases, devices disclosed herein are manufactured with acombination of materials listed herein or any other suitable materialknown in the art.

Devices described herein may comprise material having a range of tensilestrength. Exemplary materials having a range of tensile strengthsinclude, but are not limited to, nylon (70 MPa), nitrocellulose (1.5MPa), polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa),agarose (1-10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane(PDMS) (3.9-10.8 MPa). Solid supports described herein can have atensile strength from 1 to 300, 1 to 40, 1 to 10, 1 to 5, or 3 to 11MPa. Solid supports described herein can have a tensile strength ofabout 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 270, or more MPa. In some instances, adevice described herein comprises a solid support for polynucleotidesynthesis that is in the form of a flexible material capable of beingstored in a continuous loop or reel, such as a tape or flexible sheet.

Young's modulus measures the resistance of a material to elastic(recoverable) deformation under load. Exemplary materials having a rangeof Young's modulus stiffness include, but are not limited to, nylon (3GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicon (150GPa), polystyrene (3 GPa), agarose (1-10 GPa), polyacrylamide (1-10GPa), polydimethylsiloxane (PDMS) (1-10 GPa). Solid supports describedherein can have a Young's moduli from 1 to 500, 1 to 40, 1 to 10, 1 to5, or 3 to 11 GPa. Solid supports described herein can have a Young'smoduli of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa, or more. As therelationship between flexibility and stiffness are inverse to eachother, a flexible material has a low Young's modulus and changes itsshape considerably under load. In some instances, a solid supportdescribed herein has a surface with a flexibility of at least nylon.

In some cases, devices disclosed herein comprise a silicon dioxide baseand a surface layer of silicon oxide. Alternatively, the devices mayhave a base of silicon oxide. Surface of the devices provided here maybe textured, resulting in an increase overall surface area forpolynucleotide synthesis. Devices disclosed herein in some instancescomprise at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon.Devices disclosed herein in some instances are fabricated from siliconon insulator (SOI) wafer.

The structure may be fabricated from a variety of materials, suitablefor the methods and compositions of the invention described herein. Ininstances, the materials from which the substrates/solid supports of thecomprising the invention are fabricated exhibit a low level ofpolynucleotide binding. In some situations, material that aretransparent to visible and/or UV light can be employed. Materials thatare sufficiently conductive, e.g. those that can form uniform electricfields across all or a portion of the substrates/solids supportdescribed herein, can be utilized. In some instances, such materials maybe connected to an electric ground. In some cases, the substrate orsolid support can be heat conductive or insulated. The materials can bechemical resistant and heat resistant to support chemical or biochemicalreactions such as a series of polynucleotide synthesis reactions. Forflexible materials, materials of interest can include: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like.

For rigid materials, specific materials of interest include: glass; fusesilica; silicon, plastics (for example polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). The structurecan be fabricated from a material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, polydimethylsiloxane (PDMS), and glass. Thesubstrates/solid supports or the microstructures, reactors therein maybe manufactured with a combination of materials listed herein or anyother suitable material known in the art.

In some instances, a substrate disclosed herein comprises a computerreadable material. Computer readable materials include, withoutlimitation, magnetic media, reel-to-reel tape, cartridge tape, cassettetape, flexible disk, paper media, film, microfiche, continuous tape(e.g., a belt) and any media suitable for storing electronicinstructions. In some cases, the substrate comprises magneticreel-to-reel tape or a magnetic belt. In some instances, the substratecomprises a flexible printed circuit board.

Structures described herein may be transparent to visible and/or UVlight. In some instances, structures described herein are sufficientlyconductive to form uniform electric fields across all or a portion of astructure. In some instances, structures described herein are heatconductive or insulated. In some instances, the structures are chemicalresistant and heat resistant to support a chemical reaction such as apolynucleotide synthesis reaction. In some instances, the substrate ismagnetic. In some instances, the structures comprise a metal or a metalalloy.

Structures for polynucleotide synthesis may be over 1, 2, 5, 10, 30, 50or more feet long in any dimension. In the case of a flexible structure,the flexible structure is optionally stored in a wound state, e.g., in areel. In the case of a large rigid structure, e.g., greater than 1 footin length, the rigid structure can be stored vertically or horizontally.

Surface Preparation

Provided herein are methods to support the immobilization of abiomolecule on a substrate, where a surface of a structure describedherein comprises a material and/or is coated with a material thatfacilitates a coupling reaction with the biomolecule for attachment. Toprepare a structure for biomolecule immobilization, surfacemodifications may be employed that chemically and/or physically alterthe substrate surface by an additive or subtractive process to changeone or more chemical and/or physical properties of a substrate surfaceor a selected site or region of the surface. For example, surfacemodification involves (1) changing the wetting properties of a surface,(2) functionalizing a surface, i.e. providing, modifying or substitutingsurface functional groups, (3) defunctionalizing a surface, i.e.removing surface functional groups, (4) otherwise altering the chemicalcomposition of a surface, e.g., through etching, (5) increasing ordecreasing surface roughness, (6) providing a coating on a surface,e.g., a coating that exhibits wetting properties that are different fromthe wetting properties of the surface, and/or (7) depositingparticulates on a surface. In some instances, the surface of a structureis selectively functionalized to produce two or more distinct areas on astructure, wherein at least one area has a different surface or chemicalproperty that another area of the same structure. Such propertiesinclude, without limitation, surface energy, chemical termination,surface concentration of a chemical moiety, and the like.

In some instances, a surface of a structure disclosed herein is modifiedto comprise one or more actively functionalized surfaces configured tobind to both the surface of the substrate and a biomolecule, therebysupporting a coupling reaction to the surface. In some instances, thesurface is also functionalized with a passive material that does notefficiently bind the biomolecule, thereby preventing biomoleculeattachment at sites where the passive functionalization agent is bound.In some cases, the surface comprises an active layer only definingdistinct loci for biomolecule support.

In some instances, the surface is contacted with a mixture offunctionalization groups which are in any different ratio. In someinstances, a mixture comprises at least 2, 3, 4, 5 or more differenttypes of functionalization agents. In some cases, the ratio of the atleast two types of surface functionalization agents in a mixture isabout 1:1, 1:2, 1:5, 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10,9:10, or any other ratio to achieve a desired surface representation oftwo groups. In some instances, desired surface tensions, wettabilities,water contact angles, and/or contact angles for other suitable solventsare achieved by providing a substrate surface with a suitable ratio offunctionalization agents. In some cases, the agents in a mixture arechosen from suitable reactive and inert moieties, thus diluting thesurface density of reactive groups to a desired level for downstreamreactions. In some instances, the mixture of functionalization reagentscomprises one or more reagents that bind to a biomolecule and one ormore reagents that do not bind to a biomolecule. Therefore, modulationof the reagents allows for the control of the amount of biomoleculebinding that occurs at a distinct area of functionalization.

In some instances, a method for substrate functionalization comprisesdeposition of a silane molecule onto a surface of a substrate. Thesilane molecule may be deposited on a high energy surface of thesubstrate. In some instances the high surface energy region includes apassive functionalization reagent. Methods described herein provide fora silane group to bind the surface, while the rest of the moleculeprovides a distance from the surface and a free hydroxyl group at theend to which a biomolecule attaches. In some instances, the silane is anorganofunctional alkoxysilane molecule. Non-limiting examples oforganofunctional alkoxysilane molecules includedimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane,trichloro-octodecyl-silane, and trimethyl-octodecyl-silane,triethyl-octodecyl-silane. In some instances, the silane is an aminosilane. Examples of amino silanes include, without limitation,11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane,(3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane,glycidyloxypropyl/trimethoxysilane andN-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In some instances, thesilane comprises 11-acetoxyundecyltriethoxysilane,n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane,(3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane,N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, or any combinationthereof. In some instances, an active functionalization agent comprises11-acetoxyundecyltriethoxysilane. In some instances, an activefunctionalization agent comprises n-decyltriethoxysilane. In some cases,an active functionalization agent comprisesglycidyloxypropyltriethoxysilane (GOPS). In some instances, the silaneis a fluorosilane. In some instances, the silane is a hydrocarbonsilane. In some cases, the silane is 3-iodo-propyltrimethoxysilane. Insome cases, the silane is octylchlorosilane.

In some instances, silanization is performed on a surface throughself-assembly with organofunctional alkoxysilane molecules. Theorganofunctional alkoxysilanes are classified according to their organicfunctions. Non-limiting examples of siloxane functionalizing reagentsinclude hydroxyalkyl siloxanes (silylate surface, functionalizing withdiborane and oxidizing the alcohol by hydrogen peroxide), diol(dihydroxyalkyl) siloxanes (silylate surface, and hydrolyzing to diol),aminoalkyl siloxanes (amines require no intermediate functionalizingstep), glycidoxysilanes (3-glycidoxypropyl-dimethyl-ethoxysilane,glycidoxy-trimethoxysilane), mercaptosilanes(3-mercaptopropyl-trimethoxysilane, 3-4epoxycyclohexyl-ethyltrimethoxysilane or3-mercaptopropyl-methyl-dimethoxysilane),bicyclohepthenyl-trichlorosilane, butyl-aldehydr-trimethoxysilane, ordimeric secondary aminoalkyl siloxanes. Exemplary hydroxyalkyl siloxanesinclude allyl trichlorochlorosilane turning into 3-hydroxypropyl, or7-oct-1-enyl trichlorochlorosilane turning into 8-hydroxyoctyl. The diol(dihydroxyalkyl) siloxanes include glycidyl trimethoxysilane-derived(2,3-dihydroxypropyloxy)propyl (GOPS). The aminoalkyl siloxanes include3-aminopropyl trimethoxysilane turning into 3-aminopropyl(3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane,3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane).In some cases, the dimeric secondary aminoalkyl siloxanes isbis(3-trimethoxysilylpropyl) amine turning intobis(silyloxylpropyl)amine.

Active functionalization areas may comprise one or more differentspecies of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moresilanes. In some cases, one of the one or more silanes is present in thefunctionalization composition in an amount greater than another silane.For example, a mixed silane solution having two silanes comprises a99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11,88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 75:25,70:30, 65:35, 60:40, 55:45 ratio of one silane to another silane. Insome instances, an active functionalization agent comprises11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane. In someinstances, an active functionalization agent comprises11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane in a ratiofrom about 20:80 to about 1:99, or about 10:90 to about 2:98, or about5:95.

In some instances, functionalization comprises deposition of afunctionalization agent to a structure by any deposition technique,including, but not limiting to, chemical vapor deposition (CVD), atomiclayer deposition (ALD), plasma enhanced CVD (PECVD), plasma enhanced ALD(PEALD), metal organic CVD (MOCVD), hot wire CVD (HWCVD), initiated CVD(iCVD), modified CVD (MCVD), vapor axial deposition (VAD), outside vapordeposition (OVD), physical vapor deposition (e.g., sputter deposition,evaporative deposition), and molecular layer deposition (MLD).

Any step or component in the following functionalization process beomitted or changed in accordance with properties desired of the finalfunctionalized substrate. In some cases, additional components and/orprocess steps are added to the process workflows embodied herein. Insome instances, a substrate is first cleaned, for example, using apiranha solution. An example of a cleaning process includes soaking asubstrate in a piranha solution (e.g., 90% H₂SO₄, 10% H₂O₂) at anelevated temperature (e.g., 120° C.) and washing (e.g., water) anddrying the substrate (e.g., nitrogen gas). The process optionallyincludes a post piranha treatment comprising soaking the piranha treatedsubstrate in a basic solution (e.g., NH₄OH) followed by an aqueous wash(e.g., water). In some instances, a surface of a structure is plasmacleaned, optionally following the piranha soak and optional post piranhatreatment. An example of a plasma cleaning process comprises an oxygenplasma etch. In some instances, the surface is deposited with an activefunctionalization agent following by vaporization. In some instances,the substrate is actively functionalized prior to cleaning, for example,by piranha treatment and/or plasma cleaning.

The process for surface functionalization optionally comprises a resistcoat and a resist strip. In some instances, following active surfacefunctionalization, the substrate is spin coated with a resist, forexample, SPR™ 3612 positive photoresist. The process for surfacefunctionalization, in various instances, comprises lithography withpatterned functionalization. In some instances, photolithography isperformed following resist coating. In some instances, afterlithography, the surface is visually inspected for lithography defects.The process for surface functionalization, in some instances, comprisesa cleaning step, whereby residues of the substrate are removed, forexample, by plasma cleaning or etching. In some instances, the plasmacleaning step is performed at some step after the lithography step.

In some instances, a surface coated with a resist is treated to removethe resist, for example, after functionalization and/or afterlithography. In some cases, the resist is removed with a solvent, forexample, with a stripping solution comprising N-methyl-2-pyrrolidone. Insome cases, resist stripping comprises sonication or ultrasonication. Insome instances, a resist is coated and stripped, followed by activefunctionalization of the exposed areas to create a desired differentialfunctionalization pattern.

In some instances, the methods and compositions described herein relateto the application of photoresist for the generation of modified surfaceproperties in selective areas, wherein the application of thephotoresist relies on the fluidic properties of the surface defining thespatial distribution of the photoresist. Without being bound by theory,surface tension effects related to the applied fluid may define the flowof the photoresist. For example, surface tension and/or capillary actioneffects may facilitate drawing of the photoresist into small structuresin a controlled fashion before the resist solvents evaporate. In someinstances, resist contact points are pinned by sharp edges, therebycontrolling the advance of the fluid. The underlying structures may bedesigned based on the desired flow patterns that are used to applyphotoresist during the manufacturing and functionalization processes. Asolid organic layer left behind after solvents evaporate may be used topursue the subsequent steps of the manufacturing process. Structures maybe designed to control the flow of fluids by facilitating or inhibitingwicking effects into neighboring fluidic paths. For example, a structureis designed to avoid overlap between top and bottom edges, whichfacilitates the keeping of the fluid in top structures allowing for aparticular disposition of the resist. In an alternative example, the topand bottom edges overlap, leading to the wicking of the applied fluidinto bottom structures. Appropriate designs may be selected accordingly,depending on the desired application of the resist.

In some instances, a structure described herein has a surface thatcomprises a material having thickness of at least or at least 0.1 nm,0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm or 25 nm that comprises a reactive groupcapable of binding nucleosides. Exemplary include, without limitation,glass and silicon, such as silicon dioxide and silicon nitride. In somecases, exemplary surfaces include nylon and PMMA.

In some instances, electromagnetic radiation in the form of UV light isused for surface patterning. In some instances, a lamp is used forsurface patterning, and a mask mediates exposure locations of the UVlight to the surface. In some instances, a laser is used for surfacepatterning, and a shutter opened/closed state controls exposure of theUV light to the surface. The laser arrangement may be used incombination with a flexible structure that is capable of moving. In suchan arrangement, the coordination of laser exposure and flexiblestructure movement is used to create patterns of one or more agentshaving differing nucleoside coupling capabilities.

Described herein are surfaces for polynucleotide synthesis that arereusable. After synthesis and/or cleavage of polynucleotides, a surfacemay be bathed, washed, cleaned, baked, etched, or otherwise functionallyrestored to a condition suitable for subsequent polynucleotidesynthesis. The number of times a surface is reused and the methods forrecycling/preparing the surface for reuse vary depending on subsequentapplications. Surfaces prepared for reuse are in some instances reusedat least 1, 2, 3, 5, 10, 20, 50, 100, 1,000 or more times. In someinstances, the remaining “life” or number of times a surface is suitablefor reuse is measured or predicted.

Material Deposition Systems

In some cases, the synthesized polynucleotides are stored on thesubstrate, for example a solid support. Nucleic acid reagents may bedeposited on the substrate surface in a non-continuous, ordrop-on-demand method. Examples of such methods include theelectromechanical transfer method, electric thermal transfer method, andelectrostatic attraction method. In the electromechanical transfermethod, piezoelectric elements deformed by electrical pulses cause thedroplets to be ejected. In the electric thermal transfer method, bubblesare generated in a chamber of the device, and the expansive force of thebubbles causes the droplets to be ejected. In the electrostaticattraction method, electrostatic force of attraction is used to ejectthe droplets onto the substrate. In some cases, the drop frequency isfrom about 5 KHz to about 500 KHz; from about 5 KHz to about 100 KHz;from about 10 KHz to about 500 KHz; from about 10 KHz to about 100 KHz;or from about 50 KHz to about 500 KHz. In some cases, the frequency isless than about 500 KHz, 200 KHz, 100 KHz, or 50 KHz.

The size of the droplets dispensed correlates to the resolution of thedevice. In some instances, the devices deposit droplets of reagents atsizes from about 0.01 pl to about 20 pl, from about 0.01 pl to about 10pl, from about 0.01 pl to about 1 pl, from about 0.01 pl to about 0.5pl, from about 0.01 pl to about 0.01 pl, or from about 0.05 pl to about1 pl. In some instances, the droplet size is less than about 1 pl, 0.5pl, 0.2 pl, 0.1 pl, or 0.05 pl.

In some arrangements, the configuration of a polynucleotide synthesissystem allows for a continuous polynucleotide synthesis process thatexploits the flexibility of a substrate for traveling in a reel-to-reeltype process. This synthesis process operates in a continuous productionline manner with the substrate travelling through various stages ofpolynucleotide synthesis using one or more reels to rotate the positionof the substrate. In an exemplary instance, a polynucleotide synthesisreaction comprises rolling a substrate: through a solvent bath, beneatha deposition device for phosphoramidite deposition, through a bath ofoxidizing agent, through an acetonitrile wash bath, and through adeblock bath. Optionally, the tape is also traversed through a cappingbath. A reel-to-reel type process allows for the finished product of asubstrate comprising synthesized polynucleotides to be easily gatheredon a take-up reel, where it can be transported for further processing orstorage.

In some arrangements, polynucleotide synthesis proceeds in a continuousprocess as a continuous flexible tape is conveyed along a conveyor beltsystem. Similar to the reel-to-reel type process, polynucleotidesynthesis on a continuous tape operates in a production line manner,with the substrate travelling through various stages of polynucleotidesynthesis during conveyance. However, in a conveyor belt process, thecontinuous tape revisits a polynucleotide synthesis step without rollingand unrolling of the tape, as in a reel-to-reel process. In somearrangements, polynucleotide synthesis steps are partitioned into zonesand a continuous tape is conveyed through each zone one or more times ina cycle. For example, a polynucleotide synthesis reaction may comprise(1) conveying a substrate through a solvent bath, beneath a depositiondevice for phosphoramidite deposition, through a bath of oxidizingagent, through an acetonitrile wash bath, and through a block bath in acycle; and then (2) repeating the cycles to achieve synthesizedpolynucleotides of a predetermined length. After polynucleotidesynthesis, the flexible substrate is removed from the conveyor beltsystem and, optionally, rolled for storage. Rolling may be around areel, for storage. In some instances, a flexible substrate comprisingthermoplastic material is coated with nucleoside coupling reagent. Thecoating is patterned into loci such that each locus has diameter ofabout 10 um, with a center-to-center distance between two adjacent lociof about 21 um. In this instance, the locus size is sufficient toaccommodate a sessile drop volume of 0.2 pl during a polynucleotidesynthesis deposition step. In some cases, the locus density is about 2.2billion loci per m² (1 locus/441×10⁻¹² m²). In some cases, a 4.5 m²substrate comprise about 10 billion loci, each with a 10 um diameter.

In some arrangements, a device for application of one or more reagentsto a substrate during a synthesis reaction is configured to depositreagents and/or nucleoside monomers for nucleoside phosphoramidite basedsynthesis. Reagents for polynucleotide synthesis include reagents forpolynucleotide extension and wash buffers. As non-limiting examples, thedevice deposits cleaning reagents, coupling reagents, capping reagents,oxidizers, de-blocking agents, acetonitrile, gases such as nitrogen gas,and any combination thereof. In addition, the device optionally depositsreagents for the preparation and/or maintenance of substrate integrity.In some instances, the polynucleotide synthesizer deposits a drop havinga diameter less than about 200 um, 100 um, or 50 um in a volume lessthan about 1000, 500, 100, 50, or 20 pl. In some cases, thepolynucleotide synthesizer deposits between about 1 and 10000, 1 and5000, 100 and 5000, or 1000 and 5000 droplets per second.

Described herein are devices, methods, systems and compositions wherereagents for polynucleotide synthesis are recycled or reused. Recyclingof reagents may comprise collection, storage, and usage of unusedreagents, or purification/transformation of used reagents. For example,a reagent bath is recycled and used for a polynucleotide synthesis stepon the same or a different surface. Reagents described herein may berecycled 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Alternatively orin combination, a reagent solution comprising a reaction byproduct isfiltered to remove the byproduct, and the reagent solution is used foradditional polynucleotide synthesis reactions.

Many integrated or non-integrated elements are often used withpolynucleotide synthesis systems. In some instances, a polynucleotidesynthesis system comprises one or more elements useful for downstreamprocessing of synthesized polynucleotides. As an example, the systemcomprises a temperature control element such as a thermal cyclingdevice. In some instances, the temperature control element is used witha plurality of resolved reactors to perform nucleic acid assembly suchas PCA and/or nucleic acid amplification such as PCR.

De Novo Polynucleotide Synthesis

Provided herein are systems and methods for synthesis of a high densityof polynucleotides on a substrate in a short amount of time. In someinstances, the substrate is a flexible substrate. In some instances, atleast 10¹⁰, 10¹1, 10¹², 10¹³, 10¹⁴, or 10¹⁵ bases are synthesized in oneday. In some instances, at least 10×10⁸, 10×10⁹, 10×10¹⁰, 10×10¹¹, or10×10¹² polynucleotides are synthesized in one day. In some cases, eachpolynucleotide synthesized comprises at least 20, 50, 100, 200, 300, 400or 500 nucleobases. In some cases, these bases are synthesized with atotal average error rate of less than about 1 in 100; 200; 300; 400;500; 1000; 2000; 5000; 10000; 15000; 20000 bases. In some instances,these error rates are for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, 99.5%, or more of the polynucleotides synthesized. In someinstances, these at least 90%, 95%, 98%, 99%, 99.5%, or more of thepolynucleotides synthesized do not differ from a predetermined sequencefor which they encode. In some instances, the error rate for synthesizedpolynucleotides on a substrate using the methods and systems describedherein is less than about 1 in 200. In some instances, the error ratefor synthesized polynucleotides on a substrate using the methods andsystems described herein is less than about 1 in 1,000. In someinstances, the error rate for synthesized polynucleotides on a substrateusing the methods and systems described herein is less than about 1 in2,000. In some instances, the error rate for synthesized polynucleotideson a substrate using the methods and systems described herein is lessthan about 1 in 3,000. In some instances, the error rate for synthesizedpolynucleotides on a substrate using the methods and systems describedherein is less than about 1 in 5,000. Individual types of error ratesinclude mismatches, deletions, insertions, and/or substitutions for thepolynucleotides synthesized on the substrate. The term “error rate”refers to a comparison of the collective amount of synthesizedpolynucleotide to an aggregate of predetermined polynucleotidesequences. In some instances, synthesized polynucleotides disclosedherein comprise a tether of 12 to 25 bases. In some instances, thetether comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 or more bases.

Described herein are methods, systems, devices, and compositions whereinchemical reactions used in polynucleotide synthesis are controlled usingelectrochemistry. Electrochemical reactions in some instances arecontrolled by any source of energy, such as light, heat, radiation, orelectricity. For example, electrodes are used to control chemicalreactions as all or a portion of discrete loci on a surface. Electrodesin some instances are charged by applying an electrical potential to theelectrode to control one or more chemical steps in polynucleotidesynthesis. In some instances, these electrodes are addressable. Anynumber of the chemical steps described herein is in some instancescontrolled with one or more electrodes. Electrochemical reactions maycomprise oxidations, reductions, acid/base chemistry, or other reactionthat is controlled by an electrode. In some instances, electrodesgenerate electrons or protons that are used as reagents for chemicaltransformations. Electrodes in some instances directly generate areagent such as an acid. In some instances, an acid is a proton.Electrodes in some instances directly generate a reagent such as a base.Acids or bases are often used to cleave protecting groups, or influencethe kinetics of various polynucleotide synthesis reactions, for exampleby adjusting the pH of a reaction solution. Electrochemically controlledpolynucleotide synthesis reactions in some instances compriseredox-active metals or other redox-active organic materials. In someinstances, metal or organic catalysts are employed with theseelectrochemical reactions. In some instances, acids are generated fromoxidation of quinones.

Control of chemical reactions with is not limited to the electrochemicalgeneration of reagents; chemical reactivity may be influenced indirectlythrough biophysical changes to substrates or reagents through electricfields (or gradients) which are generated by electrodes. In someinstances, substrates include but are not limited to nucleic acids. Insome instances, electrical fields which repel or attract specificreagents or substrates towards or away from an electrode or surface aregenerated. Such fields in some instances are generated by application ofan electrical potential to one or more electrodes. For example,negatively charged nucleic acids are repelled from negatively chargedelectrode surfaces. Such repulsions or attractions of polynucleotides orother reagents caused by local electric fields in some instancesprovides for movement of polynucleotides or other reagents in or out ofregion of the synthesis device or structure. In some instances,electrodes generate electric fields which repel polynucleotides awayfrom a synthesis surface, structure, or device. In some instances,electrodes generate electric fields which attract polynucleotidestowards a synthesis surface, structure, or device. In some instances,protons are repelled from a positively charged surface to limit contactof protons with substrates or portions thereof. In some instances,repulsion or attractive forces are used to allow or block entry ofreagents or substrates to specific areas of the synthesis surface. Insome instances, nucleoside monomers are prevented from contacting apolynucleotide chain by application of an electric field in the vicinityof one or both components. Such arrangements allow gating of specificreagents, which may obviate the need for protecting groups when theconcentration or rate of contact between reagents and/or substrates iscontrolled. In some instances, unprotected nucleoside monomers are usedfor polynucleotide synthesis. Alternatively, application of the field inthe vicinity of one or both components promotes contact of nucleosidemonomers with a polynucleotide chain. Additionally, application ofelectric fields to a substrate can alter the substrates reactivity orconformation. In an exemplary application, electric fields generated byelectrodes are used to prevent polynucleotides at adjacent loci frominteracting. In some instances, the substrate is a polynucleotide,optionally attached to a surface. Application of an electric field insome instances alters the three-dimensional structure of apolynucleotide. Such alterations comprise folding or unfolding ofvarious structures, such as helices, hairpins, loops, or other3-dimensional nucleic acid structure. Such alterations are useful formanipulating nucleic acids inside of wells, channels, or otherstructures. In some instances, electric fields are applied to a nucleicacid substrate to prevent secondary structures. In some instances,electric fields obviate the need for linkers or attachment to a solidsupport during polynucleotide synthesis.

A suitable method for polynucleotide synthesis on a substrate of thisdisclosure is a phosphoramidite method comprising the controlledaddition of a phosphoramidite building block, i.e. nucleosidephosphoramidite, to a growing polynucleotide chain in a coupling stepthat forms a phosphite triester linkage between the phosphoramiditebuilding block and a nucleoside bound to the substrate. In someinstances, the nucleoside phosphoramidite is provided to the substrateactivated. In some instances, the nucleoside phosphoramidite is providedto the substrate with an activator. In some instances, nucleosidephosphoramidites are provided to the substrate in a 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100-fold excess or more over the substrate-boundnucleosides. In some instances, the addition of nucleosidephosphoramidite is performed in an anhydrous environment, for example,in anhydrous acetonitrile. Following addition and linkage of anucleoside phosphoramidite in the coupling step, the substrate isoptionally washed. In some instances, the coupling step is repeated oneor more additional times, optionally with a wash step between nucleosidephosphoramidite additions to the substrate. In some instances, apolynucleotide synthesis method used herein comprises 1, 2, 3 or moresequential coupling steps. Prior to coupling, in many cases, thenucleoside bound to the substrate is de-protected by removal of aprotecting group, where the protecting group functions to preventpolymerization. Protecting groups may comprise any chemical group thatprevents extension of the polynucleotide chain. In some instances, theprotecting group is cleaved (or removed) in the presence of an acid. Insome instances, the protecting group is cleaved in the presence of abase. In some instances, the protecting group is removed withelectromagnetic radiation such as light, heat, or other energy source.In some instances, the protecting group is removed through an oxidationor reduction reaction. In some instances, a protecting group comprises atriarylmethyl group. In some instances, a protecting group comprises anaryl ether. In some instances, a protecting comprises a disulfide. Insome instances a protecting group comprises an acid-labile silane. Insome instances, a protecting group comprises an acetal. In someinstances, a protecting group comprises a ketal. In some instances, aprotecting group comprises an enol ether. In some instances, aprotecting group comprises a methoxybenzyl group. In some instances, aprotecting group comprises an azide. In some instances, a protectinggroup is 4,4′-dimethoxytrityl (DMT). In some instances, a protectinggroup is a tert-butyl carbonate. In some instances, a protecting groupis a tert-butyl ester. In some instances, a protecting group comprises abase-labile group.

Following coupling, phosphoramidite polynucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingpolynucleotide is treated with a capping agent. A capping step generallyserves to block unreacted substrate-bound 5′-OH groups after couplingfrom further chain elongation, preventing the formation ofpolynucleotides with internal base deletions. Further, phosphoramiditesactivated with 1H-tetrazole often react, to a small extent, with the O6position of guanosine. Without being bound by theory, upon oxidationwith I₂/water, this side product, possibly via O6-N7 migration,undergoes depurination. The apurinic sites can end up being cleaved inthe course of the final deprotection of the polynucleotide thus reducingthe yield of the full-length product. The O6 modifications may beremoved by treatment with the capping reagent prior to oxidation withI₂/water. In some instances, inclusion of a capping step duringpolynucleotide synthesis decreases the error rate as compared tosynthesis without capping. As an example, the capping step comprisestreating the substrate-bound polynucleotide with a mixture of aceticanhydride and 1-methylimidazole. Following a capping step, the substrateis optionally washed.

Following addition of a nucleoside phosphoramidite, and optionally aftercapping and one or more wash steps, a substrate described hereincomprises a bound growing nucleic acid that may be oxidized. Theoxidation step comprises oxidizing the phosphite triester into atetracoordinated phosphate triester, a protected precursor of thenaturally occurring phosphate diester internucleoside linkage. In someinstances, phosphite triesters are oxidized electrochemically. In someinstances, oxidation of the growing polynucleotide is achieved bytreatment with iodine and water, optionally in the presence of a weakbase such as a pyridine, lutidine, or collidine. Oxidation is sometimescarried out under anhydrous conditions using tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for substrate drying, as residual water from oxidation that maypersist can inhibit subsequent coupling. Following oxidation, thesubstrate and growing polynucleotide is optionally washed. In someinstances, the step of oxidation is substituted with a sulfurizationstep to obtain polynucleotide phosphorothioates, wherein any cappingsteps can be performed after the sulfurization. Many reagents arecapable of the efficient sulfur transfer, including, but not limited to,3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

For a subsequent cycle of nucleoside incorporation to occur throughcoupling, a protected 5′ end (or 3′ end, if synthesis is conducted in a5′ to 3′ direction) of the substrate bound growing polynucleotide is beremoved so that the primary hydroxyl group can react with a nextnucleoside phosphoramidite. In some instances, the protecting group isDMT and deblocking occurs with trichloroacetic acid in dichloromethane.In some instances, the protecting group is DMT and deblocking occurswith electrochemically generated protons. Conducting detritylation foran extended time or with stronger than recommended solutions of acidsmay lead to increased depurination of solid support-bound polynucleotideand thus reduces the yield of the desired full-length product. Methodsand compositions described herein provide for controlled deblockingconditions limiting undesired depurination reactions. In some instances,the substrate bound polynucleotide is washed after deblocking. In somecases, efficient washing after deblocking contributes to synthesizedpolynucleotides having a low error rate.

Methods for the synthesis of polynucleotides on a substrate describedherein may involve an iterating sequence of the following steps:application of a protected monomer to a surface of a substrate featureto link with either the surface, a linker or with a previouslydeprotected monomer; deprotection of the applied monomer so that it canreact with a subsequently applied protected monomer; and application ofanother protected monomer for linking. One or more intermediate stepsinclude oxidation and/or sulfurization. In some instances, one or morewash steps precede or follow one or all of the steps.

Methods for the synthesis of polynucleotides on a substrate describedherein may comprise an oxidation step. For example, methods involve aniterating sequence of the following steps: application of a protectedmonomer to a surface of a substrate feature to link with either thesurface, a linker or with a previously deprotected monomer; deprotectionof the applied monomer so that it can react with a subsequently appliedprotected monomer; application of another protected monomer for linking,and oxidation and/or sulfurization. In some instances, one or more washsteps precede or follow one or all of the steps.

Methods for the synthesis of polynucleotides on a substrate describedherein may further comprise an iterating sequence of the followingsteps: application of a protected monomer to a surface of a substratefeature to link with either the surface, a linker or with a previouslydeprotected monomer; deprotection of the applied monomer so that it canreact with a subsequently applied protected monomer; and oxidationand/or sulfurization. In some instances, one or more wash steps precedeor follow one or all of the steps.

Methods for the synthesis of polynucleotides on a substrate describedherein may further comprise an iterating sequence of the followingsteps: application of a protected monomer to a surface of a substratefeature to link with either the surface, a linker or with a previouslydeprotected monomer; and oxidation and/or sulfurization. In someinstances, one or more wash steps precede or follow one or all of thesteps.

Methods for the synthesis of polynucleotides on a substrate describedherein may further comprise an iterating sequence of the followingsteps: application of a protected monomer to a surface of a substratefeature to link with either the surface, a linker or with a previouslydeprotected monomer; deprotection of the applied monomer so that it canreact with a subsequently applied protected monomer; and oxidationand/or sulfurization. In some instances, one or more wash steps precedeor follow one or all of the steps.

In some instances, polynucleotides are synthesized with photolabileprotecting groups, where the hydroxyl groups generated on the surfaceare blocked by photolabile-protecting groups. When the surface isexposed to UV light, such as through a photolithographic mask, a patternof free hydroxyl groups on the surface may be generated. These hydroxylgroups can react with photoprotected nucleoside phosphoramidites,according to phosphoramidite chemistry. A second photolithographic maskcan be applied and the surface can be exposed to UV light to generatesecond pattern of hydroxyl groups, followed by coupling with5′-photoprotected nucleoside phosphoramidite. Likewise, patterns can begenerated and oligomer chains can be extended. Without being bound bytheory, the lability of a photocleavable group depends on the wavelengthand polarity of a solvent employed and the rate of photocleavage may beaffected by the duration of exposure and the intensity of light. Thismethod can leverage a number of factors such as accuracy in alignment ofthe masks, efficiency of removal of photo-protecting groups, and theyields of the phosphoramidite coupling step. Further, unintended leakageof light into neighboring sites can be minimized. The density ofsynthesized oligomer per spot can be monitored by adjusting loading ofthe leader nucleoside on the surface of synthesis.

The surface of a substrate described herein that provides support forpolynucleotide synthesis may be chemically modified to allow for thesynthesized polynucleotide chain to be cleaved from the surface. In someinstances, the polynucleotide chain is cleaved at the same time as thepolynucleotide is deprotected. In some cases, the polynucleotide chainis cleaved after the polynucleotide is deprotected. In an exemplaryscheme, a trialkoxysilyl amine such as (CH₃CH₂O)₃Si—(CH₂)₂—NH₂ isreacted with surface SiOH groups of a substrate, followed by reactionwith succinic anhydride with the amine to create an amide linkage and afree OH on which the nucleic acid chain growth is supported. Cleavageincludes gas cleavage with ammonia or methylamine. In some instancescleavage includes linker cleavage with electrically generated reagentssuch as acids or bases. In some instances, once released from thesurface, polynucleotides are assembled into larger nucleic acids thatare sequenced and decoded to extract stored information.

The surfaces described herein can be reused after polynucleotidecleavage to support additional cycles of polynucleotide synthesis. Forexample, the linker can be reused without additional treatment/chemicalmodifications. In some instances, a linker is non-covalently bound to asubstrate surface or a polynucleotide. In some embodiments, the linkerremains attached to the polynucleotide after cleavage from the surface.Linkers in some embodiments comprise reversible covalent bonds such asesters, amides, ketals, beta substituted ketones, heterocycles, or othergroup that is capable of being reversibly cleaved. Such reversiblecleavage reactions are in some instances controlled through the additionor removal of reagents, or by electrochemical processes controlled byelectrodes. Optionally, chemical linkers or surface-bound chemicalgroups are regenerated after a number of cycles, to restore reactivityand remove unwanted side product formation on such linkers orsurface-bound chemical groups.

Assembly

Polynucleotides may be designed to collectively span a large region of apredetermined sequence that encodes for information. In some instances,larger polynucleotides are generated through ligation reactions to jointhe synthesized polynucleotides. One example of a ligation reaction ispolymerase chain assembly (PCA). In some instances, at least of aportion of the polynucleotides are designed to include an appendedregion that is a substrate for universal primer binding. For PCAreactions, the presynthesized polynucleotides include overlaps with eachother (e.g., 4, 20, 40 or more bases with overlapping sequence). Duringthe polymerase cycles, the polynucleotides anneal to complementaryfragments and then are filled in by polymerase. Each cycle thusincreases the length of various fragments randomly depending on whichpolynucleotides find each other. Complementarity amongst the fragmentsallows for forming a complete large span of double-stranded DNA. In somecases, after the PCA reaction is complete, an error correction step isconducted using mismatch repair detecting enzymes to remove mismatchesin the sequence. Once larger fragments of a target sequence aregenerated, they can be amplified. For example, in some cases, a targetsequence comprising 5′ and 3′ terminal adapter sequences is amplified ina polymerase chain reaction (PCR) which includes modified primers thathybridize to the adapter sequences. In some cases, the modified primerscomprise one or more uracil bases. The use of modified primers allowsfor removal of the primers through enzymatic reactions centered ontargeting the modified base and/or gaps left by enzymes which cleave themodified base pair from the fragment. What remains is a double-strandedamplification product that lacks remnants of adapter sequence. In thisway, multiple amplification products can be generated in parallel withthe same set of primers to generate different fragments ofdouble-stranded DNA.

Error correction may be performed on synthesized polynucleotides and/orassembled products. An example strategy for error correction involvessite-directed mutagenesis by overlap extension PCR to correct errors,which is optionally coupled with two or more rounds of cloning andsequencing. In certain instances, double-stranded nucleic acids withmismatches, bulges and small loops, chemically altered bases and/orother heteroduplexes are selectively removed from populations ofcorrectly synthesized nucleic acids. In some instances, error correctionis performed using proteins/enzymes that recognize and bind to or nextto mismatched or unpaired bases within double-stranded nucleic acids tocreate a single or double-strand break or to initiate a strand transfertransposition event. Non-limiting examples of proteins/enzymes for errorcorrection include endonucleases (T7 Endonuclease I, E. coliEndonuclease V, T4 Endonuclease VII, mung bean nuclease, Cell, E. coliEndonuclease IV, UVDE), restriction enzymes, glycosylases,ribonucleases, mismatch repair enzymes, resolvases, helicases, ligases,antibodies specific for mismatches, and their variants. Examples ofspecific error correction enzymes include T4 endonuclease 7, T7endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH, MutL,cleavase, CELI, and HINF1. In some cases, DNA mismatch-binding proteinMutS (Therms aquaticus) is used to remove failure products from apopulation of synthesized products. In some instances, error correctionis performed using the enzyme Correctase. In some cases, errorcorrection is performed using SURVEYOR endonuclease (Transgenomic), amismatch-specific DNA endonuclease that scans for known and unknownmutations and polymorphisms for heteroduplex DNA.

Sequencing

After extraction and/or amplification of polynucleotides from thesurface of the structure, suitable sequencing technology may be employedto sequence the polynucleotides. In some cases, the DNA sequence is readon the substrate or within a feature of a structure. In some cases, thepolynucleotides stored on the substrate are extracted is optionallyassembled into longer nucleic acids and then sequenced.

Polynucleotides synthesized and stored on the structures describedherein encode data that can be interpreted by reading the sequence ofthe synthesized polynucleotides and converting the sequence into binarycode readable by a computer. In some cases the sequences requireassembly, and the assembly step may need to be at the nucleic acidsequence stage or at the digital sequence stage.

Provided herein are detection systems comprising a device capable ofsequencing stored polynucleotides, either directly on the structureand/or after removal from the main structure. In cases where thestructure is a reel-to-reel tape of flexible material, the detectionsystem comprises a device for holding and advancing the structurethrough a detection location and a detector disposed proximate thedetection location for detecting a signal originated from a section ofthe tape when the section is at the detection location. In someinstances, the signal is indicative of a presence of a polynucleotide.In some instances, the signal is indicative of a sequence of apolynucleotide (e.g., a fluorescent signal). In some instances,information encoded within polynucleotides on a continuous tape is readby a computer as the tape is conveyed continuously through a detectoroperably connected to the computer. In some instances, a detectionsystem comprises a computer system comprising a polynucleotidesequencing device, a database for storage and retrieval of data relatingto polynucleotide sequence, software for converting DNA code of apolynucleotide sequence to binary code, a computer for reading thebinary code, or any combination thereof.

Provided herein are sequencing systems that can be integrated into thedevices described herein. Various methods of sequencing are well knownin the art, and comprise “base calling” wherein the identity of a basein the target polynucleotide is identified. In some instances,polynucleotides synthesized using the methods, devices, compositions,and systems described herein are sequenced after cleavage from thesynthesis surface. In some instances, sequencing occurs during orsimultaneously with polynucleotide synthesis, wherein base callingoccurs immediately after or before extension of a nucleoside monomerinto the growing polynucleotide chain. Methods for base calling includemeasurement of electrical currents generated by polymerase-catalyzedaddition of bases to a template strand. In some instances, synthesissurfaces comprise enzymes, such as polymerases. In some instances, suchenzymes are tethered to electrodes or to the synthesis surface.

Computer Systems

In various aspects, any of the systems described herein are operablylinked to a computer and are optionally automated through a computereither locally or remotely. In various instances, the methods andsystems of the invention further comprise software programs on computersystems and use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of theinvention. In some instances, the computer systems are programmed tointerface between the user specified base sequence and the position of amaterial deposition device to deliver the correct reagents to specifiedregions of the substrate.

The computer system 800 illustrated in FIG. 8 may be understood as alogical apparatus that can read instructions from media 811 and/or anetwork port 805, which can optionally be connected to server 809 havingfixed media 812. The system can include a CPU 801, disk drives 803,optional input devices such as keyboard 815 and/or mouse 816 andoptional monitor 807. Data communication can be achieved through theindicated communication medium to a server at a local or a remotelocation. The communication medium can include any means of transmittingand/or receiving data. For example, the communication medium can be anetwork connection, a wireless connection or an internet connection.Such a connection can provide for communication over the World Wide Web.It is envisioned that data relating to the present disclosure can betransmitted over such networks or connections for reception and/orreview by a party 822.

FIG. 9 is a block diagram illustrating a first example architecture of acomputer system that can be used in connection with example instances ofthe present invention. As depicted in FIG. 5, the example computersystem can include a processor 902 for processing instructions.Non-limiting examples of processors include: Intel Xeon™ processor, AMDOpteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor,ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™processor, Marvell PXA 930™ processor, or a functionally-equivalentprocessor. Multiple threads of execution can be used for parallelprocessing. In some instances, multiple processors or processors withmultiple cores can also be used, whether in a single computer system, ina cluster, or distributed across systems over a network comprising aplurality of computers, cell phones, and/or personal data assistantdevices.

As illustrated in FIG. 9, a high speed cache 904 can be connected to, orincorporated in, the processor 902 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 902. The processor 902 is connected to a north bridge 906 by aprocessor bus 908. The north bridge 906 is connected to random accessmemory (RAM) 910 by a memory bus 912 and manages access to the RAM 910by the processor 902. The north bridge 906 is also connected to a southbridge 914 by a chipset bus 916. The south bridge 914 is, in turn,connected to a peripheral bus 918. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 918. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip.

In some instances, a system 900 can include an accelerator card 922attached to the peripheral bus 918. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing. For example, an accelerator can be used for adaptivedata restructuring or to evaluate algebraic expressions used in extendedset processing.

Software and data are stored in external storage 924 and can be loadedinto RAM 910 and/or cache 904 for use by the processor. The system 900includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MACOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample embodiments of the present invention.

In this example, system 900 also includes network interface cards (NICs)920 and 921 connected to the peripheral bus for providing networkinterfaces to external storage, such as Network Attached Storage (NAS)and other computer systems that can be used for distributed parallelprocessing.

FIG. 10 is a diagram showing a network 1000 with a plurality of computersystems 1002 a, and 1002 b, a plurality of cell phones and personal dataassistants 1002 c, and Network Attached Storage (NAS) 1004 a, and 1004b. In example embodiments, systems 1002 a, 1002 b, and 1002 c can managedata storage and optimize data access for data stored in NetworkAttached Storage (NAS) 1004 a and 1004 b. A mathematical model can beused for the data and be evaluated using distributed parallel processingacross computer systems 1002 a, and 1002 b, and cell phone and personaldata assistant systems 1002 c. Computer systems 1002 a, and 1002 b, andcell phone and personal data assistant systems 1002 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 1004 a and 1004 b. FIG. 10 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various embodiments of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some example embodiments, processors can maintain separate memoryspaces and transmit data through network interfaces, back plane or otherconnectors for parallel processing by other processors. In otherembodiments, some or all of the processors can use a shared virtualaddress memory space.

FIG. 11 is a block diagram of a multiprocessor computer system 1100using a shared virtual address memory space in accordance with anexample embodiment. The system includes a plurality of processors 1102a-f that can access a shared memory subsystem 1104. The systemincorporates a plurality of programmable hardware memory algorithmprocessors (MAPs) 1106 a-f in the memory subsystem 1104. Each MAP 1106a-f can comprise a memory 1108 a-f and one or more field programmablegate arrays (FPGAs) 1110 a-f. The MAP provides a configurable functionalunit and particular algorithms or portions of algorithms can be providedto the FPGAs 1110 a-f for processing in close coordination with arespective processor. For example, the MAPs can be used to evaluatealgebraic expressions regarding the data model and to perform adaptivedata restructuring in example embodiments. In this example, each MAP isglobally accessible by all of the processors for these purposes. In oneconfiguration, each MAP can use Direct Memory Access (DMA) to access anassociated memory 1108 a-f, allowing it to execute tasks independentlyof, and asynchronously from, the respective microprocessor 1102 a-f Inthis configuration, a MAP can feed results directly to another MAP forpipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleembodiments, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some embodiments,all or part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example embodiments, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example embodiments, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other embodiments, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs), system on chips (SOCs), application specific integratedcircuits (ASICs), or other processing and logic elements. For example,the Set Processor and Optimizer can be implemented with hardwareacceleration through the use of a hardware accelerator card.

Embodiments

Provided herein are methods for storing information, comprising: a)providing a structure comprising a surface; b) depositing at least onenucleotide on the surface, wherein the at least one nucleotide couplesto a polynucleotide attached to the surface; and c) repeating step b) tosynthesize a plurality of polynucleotides on the surface, wherein astorage density of unique polynucleotides on the surface is at least100×10⁶ polynucleotides per cm². Further provided herein are methods,wherein the method further comprises cleaving at least onepolynucleotide from the surface, wherein the polynucleotide is dissolvedin a droplet. Further provided herein are methods, wherein the methodfurther comprises sequencing at least one polynucleotide from thesurface. Further provided herein are methods, wherein the method furthercomprises drying the surface. Further provided herein are methods,wherein the method further comprises washing the nucleotides away fromthe surface. Further provided herein are methods, wherein the surface isa solid support. Further provided herein are methods, wherein thesurface comprises glass, fuse silica, silicon, silicon dioxide, siliconnitride, plastics, metals, or combinations thereof.

Provided herein are methods for storing information, comprising: a)providing a structure comprising a surface; b) depositing at dropletcomprising at least one nucleotide on the surface, wherein the at leastone nucleotide couples to a polynucleotide attached to the surface; andc) repeating step b) to synthesize a plurality of polynucleotides on thesurface, wherein the droplet has a volume of less than about 100femtoliters.

Provided herein are methods for storing information, comprising: a)providing a structure comprising a surface; b) depositing at least onenucleotide on the surface, wherein the at least one nucleotide couplesto a polynucleotide attached to the surface; and c) repeating step b) tosynthesize a plurality of polynucleotides on the surface, wherein thetime to repeat step b) using four different nucleotides is less thanabout 100 milliseconds. Further provided herein are methods, wherein themethod further comprises one or more wash steps. Further provided hereinare methods, wherein the method further comprises deblocking, oxidizing,washing, capping, or any combination thereof.

Provided herein are devices for storing information, comprising: a chipcomprising an array of addressable loci, wherein one or more addressableloci comprise at least one electrode, a synthesis surface, and at leastone fluid port, wherein the synthesis surface at each addressable locicomprises at least one polynucleotide extending from the surface,wherein a density of addressable loci on the chip is at least 100×10⁶polynucleotides per cm². Further provided herein are devices, whereinthe at least one polynucleotide is about 150 to about 500 bases inlength. Further provided herein are devices, wherein the at least onepolynucleotide are about 200 bases in length. Further provided hereinare devices, wherein the device further comprises a reagent reservoir.Further provided herein are devices, wherein the device furthercomprises a heating or cooling unit. Further provided herein aredevices, wherein at least one addressable locus comprises a droplet.Further provided herein are devices, wherein the droplet is less than 50micrometers in diameter.

Provided herein are methods for storing information, the methodcomprising: a) converting at least one item of information in a form ofat least one digital sequence to at least one nucleic acid sequence; b)synthesizing a plurality of polynucleotides having predeterminedsequences collectively encoding for the at least one nucleic acidsequence; c) depositing at droplet comprising at least one nucleotide ona surface, wherein the at least one nucleotide couples to apolynucleotide attached to the surface; d) repeating step c) tosynthesize the plurality of polynucleotides on the surface; and e)storing the plurality of polynucleotides, wherein the droplet has avolume of less than about 100 femtoliters.

Provided herein are methods for storing information, the methodcomprising: a) converting at least one item of information in a form ofat least one digital sequence to at least one nucleic acid sequence; b)synthesizing a plurality of polynucleotides having predeterminedsequences collectively encoding for the at least one nucleic acidsequence; c) depositing at droplet comprising at least one nucleotide ona surface, wherein the at least one nucleotide couples to apolynucleotide attached to the surface; d) repeating step c) tosynthesize the plurality of polynucleotides on the surface; and e)storing the plurality of polynucleotides, wherein the time to repeatstep c) using four different nucleotides is less than about 100milliseconds.

Provided herein are methods of synthesizing polynucleotides, comprising:a) providing a structure comprising a surface, wherein the surfacecomprises a plurality of loci for nucleotide extension; and b)synthesizing a plurality of polynucleotides extending from the surface,wherein synthesizing comprises depositing one or more reagents byapplying a potential to the surface. Further provided are methods,wherein the potential is an electric potential. Further provided aremethods, wherein the surface is a solid support.

Provided herein are devices for information storage using any one of themethods described herein. Provided herein are systems for informationstorage using any one of the methods described herein.

The following examples are set forth to illustrate more clearly theprinciple and practice of embodiments disclosed herein to those skilledin the art and are not to be construed as limiting the scope of anyclaimed embodiments. Unless otherwise stated, all parts and percentagesare on a weight basis.

EXAMPLES Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of alibrary of polynucleotides. The device surface was first wet cleanedusing a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20minutes. The device was rinsed in several beakers with DI water, heldunder a DI water gooseneck faucet for 5 min, and dried with N₂. Thedevice was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min,rinsed with DI water using a handgun, soaked in three successive beakerswith DI water for 1 min each, and then rinsed again with DI water usingthe handgun. The device was then plasma cleaned by exposing the devicesurface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solutioncomprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using aYES-1224P vapor deposition oven system with the following parameters:0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface wasresist coated using a Brewer Science 200× spin coater. SPR™ 3612photoresist was spin coated on the device at 2500 rpm for 40 sec. Thedevice was pre-baked for 30 min at 90° C. on a Brewer hot plate. Thedevice was subjected to photolithography using a Karl Suss MA6 maskaligner instrument. The device was exposed for 2.2 sec and developed for1 min in MSF 26A. Remaining developer was rinsed with the handgun andthe device soaked in water for 5 min. The device was baked for 30 min at100° C. in the oven, followed by visual inspection for lithographydefects using a Nikon L200. A cleaning process was used to removeresidual resist using the SAMCO PC-300 instrument to O₂ plasma etch at250 watts for 1 min.

The device surface was passively functionalized with a 100 μL solutionof perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. Thedevice was placed in a chamber, pumped for 10 min, and then the valvewas closed to the pump and left to stand for 10 min. The chamber wasvented to air. The device was resist stripped by performing two soaksfor 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power(9 on Crest system). The device was then soaked for 5 min in 500 mLisopropanol at room temperature with ultrasonication at maximum power.The device was dipped in 300 mL of 200 proof ethanol and blown dry withN₂. The functionalized surface was activated to serve as a support forpolynucleotide synthesis.

Example 2: Highly Accurate DNA-Based Information Storage and Assembly

Digital information was selected in the form of binary data totalingabout 0.2 GB included content for the Universal Declaration of HumanRights in more than 100 languages, the top 100 books of ProjectGuttenberg and a seed database. The digital information was encryptedinto a nucleic acid-based sequence and divided into strings. Over 10million non-identical polynucleotides, each corresponding to a string,were synthesized on a rigid silicon surface. Each non-identicalpolynucleotide was under equal or less than 200 bases in length. Thesynthesized polynucleotides were collected and sequenced and decodedback to digital code, with 100% accuracy for the source digitalinformation, compared to the initial at least one digital sequence.

Example 3: High Density Information Storage System

Polynucleotides are de novo synthesized by methods described herein.Following synthesis, the polynucleotides are collected into a singledroplet and transferred to storage on a silicon solid support. The solidsupport has dimensions of 86 mm×54 mm and is 1-2 mm thick. The capacityof the solid support is 1-10 petabytes (PB) that is implemented as anaddressable array of 10 gigabyte packets. The physical partitioning intopackets is redundantly encoded as a leading sequence with eachpolynucleotide within a packet sharing a common initial sequence and anyother sequence information for indexing or searching. Packets areimplemented as aqueous droplets with dissolved polynucleotides, withphysical redundancy of 100-1000 copies of each polynucleotide. Thepolynucleotide length is in a range of 100-1000 bases. Droplet volume isequivalent to spheres 40-50 μm in diameter. The solid support furthercomprises up to 10,000×10,000 positions in an area less than a squareinch.

An exemplary solid support can be seen in FIGS. 12A-12B. FIG. 12A showsa front side of the solid support made of glass and comprising a clearwindow for array and fluidic ports. FIG. 12B shows a back side of thesolid support that is a circuit comprising electrical contacts (LGA 1 mmpitch) and a thermal interface under the solid support area.

Following addition of the droplets to the solid support, the solidsupport may be dried and later resolvated for use for downstreamapplications. Alternatively, the solid support is dried and stored.Because the droplets within each packet comprise sequence informationfor indexing and searching, specific packets are retrieved from theplurality of packets based on the sequence information.

Example 4: High Density Information Storage System With Access

Polynucleotides are de novo synthesized by methods described herein.Following synthesis, the polynucleotides are collected into a singledroplet and transferred to storage on a paper solid support. The solidsupport has dimensions of 3.5 inches by 2.5 inches. The capacity of thesolid support is 1 petabyte that is implemented as an addressable 32×32array comprising 1024 spots. Each spot comprises 1 terabyte pool. Seee.g. FIG. 17 and FIG. 18.

At least one item of information of 10-100 megabytes is encoded in DNAand stored in 1 petabyte of data. At a later time, the 10-100 megabytesof encoded DNA is retrieved by random access of the encoded DNA andretrieving the encoded DNA from 1 terabyte pool.

Example 5: Local Control of Polynucleotide Synthesis on a Solid Support

Polynucleotides of 240 bases in length are synthesized on a solidsupport using the methods described herein. Polynucleotides comprisingdsDNA are approximately 80 nm in length, and polynucleotides comprisingssDNA are approximately 160 nm in length. The solid support comprises anarray of 500 nm (depth)×400 nm (diameter) wells (volume approximately0.628 femtoliters). Each wells comprises an addressable locus,addressable electrodes inside the sidewalls of each well (area is 50,000nm²/electrode), and a 250 nm (diameter) surface for polynucleotidesynthesis/attachment in addressable communication with a 250 nm(diameter) addressable bottom electrode. Electrodes are independentlyaddressable. Polynucleotides are present on the synthesis surface at adensity of 1 polynucleotide per 50 nm². Wells are separated by a pitchof 1.0 um. 10 nm thick sidewall electrodes (located about 100 nm abovethe polynucleotide surface) are charged to generate a gradient of H⁺ions that remove protecting groups (wherein the polynucleotide isblocked with an acid-cleavable blocking group) from the 5′ OH groups ondefined polynucleotides at loci on the synthesis surface. H⁺ ions arethen removed. (See FIG. 16B). A nucleoside phosphoramidite monomer isadded, and the polynucleotides at unblocked sites and will be availablefor coupling to nucleosides. Cycles of deprotection and coupling arerepeated to synthesis the polynucleotides. By applying a series ofelectrode-controlled masks to the surface before addition of each typeof monomer, the desired polynucleotides are synthesized at exactlocations on the surface in a controlled sequence.

Example 6: Local Control of Polynucleotide Synthesis on a Solid SupportWith Electric Fields

Polynucleotides of 240 bases in length are synthesized on a solidsupport using the methods described herein. The solid support comprisesan array of 500 nm (depth)×500 nm (diameter) wells. Each wells comprisesan addressable locus, addressable electrodes inside the sidewalls ofeach well (area is 50,000 nm²/electrode), and a 250 nm (diameter)surface for polynucleotide synthesis/attachment in addressablecommunication with a 250 nm (diameter) addressable bottom electrode.Electrodes are independently addressable.

Polynucleotides are present on the synthesis surface at a density of 1polynucleotide per 50 nm². Wells are separated by a pitch of 1.0 um. Anucleoside phosphoramidite monomer is added, and the polynucleotides atunblocked sites and will be available for coupling to nucleosides.Cycles of deprotection and coupling are repeated to synthesis thepolynucleotides. By applying a series of electrode-controlled masks tothe surface before addition of each type of monomer, the desiredpolynucleotides are synthesized at exact locations on the surface in acontrolled sequence. Bottom electrodes in the bottom of the well areactivated, which induces release of the polynucleotides attachedthereto. Sidewall electrodes are charged to generate an electric fieldwhich moves the polynucleotides out of the well. Nucleosidephosphoramidite monomers are then added which extend from reusablelinkers attached to the surface, and synthesis is repeated.

Example 7: Local Control of Polynucleotide Synthesis on a Solid Support

Polynucleotides of 240 bases in length are synthesized on a solidsupport using the methods described herein. Polynucleotides comprisingdsDNA are approximately 80 nm in length, and polynucleotides comprisingssDNA are approximately 160 nm in length. The solid support comprises anarray of 100 nm diameter addressable electrodes on the surface forpolynucleotide synthesis/attachment (See FIG. 19).

Polynucleotides are present on the synthesis surface at a density of 1polynucleotide per 39.27 nm². Wells are separated by a pitch of 0.25 um.The electrodes are charged to generate a gradient of H⁺ ions that removeprotecting groups (wherein the polynucleotide is blocked with anacid-cleavable blocking group) from the 5′ OH groups on definedpolynucleotides at loci on the synthesis surface. H⁺ ions are thenremoved. A nucleoside phosphoramidite monomer is added, and thepolynucleotides at unblocked sites and will be available for coupling tonucleosides. Cycles of deprotection and coupling are repeated tosynthesis the polynucleotides. By applying a series ofelectrode-controlled masks to the surface before addition of each typeof monomer, the desired polynucleotides are synthesized at exactlocations on the surface in a controlled sequence.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for storing nucleic acid-based digitalinformation, comprising: a) providing a device comprising: a solidsupport, wherein the solid support comprises a plurality of wells,wherein each of the wells comprises an addressable locus comprising: asynthesis surface located in a bottom region of each of the wells; abottom electrode in addressable communication with the synthesissurface; and at least one sidewall electrode located on a sidewall ofeach of the wells, wherein the at least one sidewall electrode is 50 nmto 200 nm from the bottom region, wherein the at least one sidewallelectrode and the bottom electrode are independently addressable; b)providing instructions for polynucleotide synthesis; c) depositing atleast one nucleoside on the synthesis surface, wherein the at least onenucleoside couples to a polynucleotide attached to the synthesissurface; d) deblocking at least one nucleoside on the synthesis surface,wherein deblocking comprises applying an electrical potential to the atleast one sidewall electrode to generate a deprotection reagent; and e)repeating steps c) and d) to synthesize a plurality of polynucleotideson the synthesis surface, wherein the instructions comprise at least onesequence encoding for the plurality of polynucleotides, wherein themethod further comprises at least one oxidation step, and wherein atleast some of the polynucleotides encode for digital information.
 2. Themethod of claim 1, wherein the solid support comprises addressable lociat a density of at least 100×10⁶ addressable loci per cm².
 3. The methodof claim 1, wherein the addressable locus comprises a diameter up toabout 750 nm.
 4. The method of claim 1, wherein the method furthercomprises cleaving at least one polynucleotide from the surface, whereinthe polynucleotide is dissolved in a droplet.
 5. The method of claim 1,wherein the method further comprises sequencing at least onepolynucleotide from the surface.
 6. The method of claim 1, wherein thenucleoside comprises a nucleoside phosphoramidite.
 7. The method ofclaim 1, wherein the method further comprises drying the surface.
 8. Themethod of claim 1, wherein the method further comprises a cleavage step,wherein the cleavage step comprises applying an electrical potential tothe bottom electrode to generate a cleavage reagent.
 9. The method ofclaim 1, wherein the method further comprises a capping step.
 10. Themethod of claim 1, wherein the method further comprises an oxidationstep between steps c) and d).
 11. The method of claim 1, wherein each ofthe wells comprises a longest cross-sectional diameter of 100 nm to 800nm.
 12. The method of claim 1, wherein the bottom electrode comprises anarea of 10⁴ nm² to 10⁵ nm².
 13. The method of claim 1, wherein at leastone of the sidewall electrodes is 75 nm to 125 nm from the bottomregion.
 14. The method of claim 1, wherein at least one of the sidewallelectrodes comprises a height of 5 nm to 25 nm.